Printed edition published in the UK in 2013 by

The Medical Research Council

Laboratory of Molecular Biology

Francis Crick Avenue,

Cambridge Biomedical Campus,

Cambridge CB2 0QH, UK

This electronic edition published in 2013 by The Medical Research Council

ISBN: 978-184831-669-0 (ePub format)

Individual articles copyright © the authors, 2013

This edition copyright © MRC-LMB, 2013

The authors have asserted their moral rights.

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Typesetting by Marie Doherty

The article by Sidney Altman was originally published in Genetics in 2003 and is reprinted with permission of the Genetic Society of America. The articles by Richard E. Dickerson and Michael G. Rossman were originally published in the Journal of Molecular Biology in 2009 and are reproduced here by permission of Elsevier.

Preface

Hugh Huxley

It is well known that during the second half of the 20th century a great revolution took place in the biological sciences, when many mysteries concerning the nature of life on Earth were shown to be capable of detailed, rational explanation. This came about through the rise of molecular biology, the understanding of biological processes in terms of the detailed molecular structures and interactions of the component molecules. It is a fact of history that a single laboratory in Cambridge, England – the Medical Research Council Laboratory of Molecular Biology (the MRC-LMB) – played a unique role in this revolution, since the scientists who founded that lab made many of the basic discoveries which opened up this field.

What is perhaps less well known is the role that this laboratory also played in helping to spread this new scientific discipline throughout the world, particularly (but not exclusively) to the United States. Beginning in the late 1950s and early 60s a growing number of visiting scientists came to work with this group, for one or two years or even longer; to learn the new techniques, to participate in the illuminating experiments now made possible, and to absorb the new concepts being developed. Many of the visitors were at the immediate postdoc stage, eager to participate in this exciting and promising new field; others were more established, but used a sabbatical year to extend their interests and expertise. Their presence greatly enhanced the lab and contributed significantly to its scientific success. In an imperfect world, it stood out as a shining example of what human intelligence, optimism, collaboration, hard work and good sense can sometimes create – to say nothing of the thirteen members of the lab who have won Nobel Prizes – one of them twice – or of six of the visitors who have won Nobel Prizes for their subsequent work.

How did it come about that so many very capable scientists worked in this one laboratory and made so many important discoveries there? This volume of memoirs by visitors to the lab may provide some clues about the culture of the lab which made it such a unique place to work. The way in which the original founders of the lab came together, and the particular problems that attracted them, may also be relevant, as I will describe briefly.

The group began to come together in Cambridge soon after the Second World War in a general atmosphere of great optimism about the future, which in this case was fully justified. Max Perutz had begun X-ray diffraction studies on the protein haemoglobin in Bernal’s group in Cambridge in 1936, believing that protein structure would be the key to many biological problems. After the war he continued this work in the Cavendish Laboratory under Sir Lawrence Bragg, who had invented the X-ray diffraction technique. John Kendrew joined him in 1946, having been advised by Bernal (during distinguished wartime service together) that this would be a particularly important field. In 1947, with Bragg’s support, Max and John persuaded the Medical Research Council to support them for five years as a small ‘Unit for Research on the Molecular Structure of Biological Systems’, with two assistants or research students, on the premise that success would be of very great value and importance, but that it was very far from being assured.

I finished my first degree in Part II Physics at the Cavendish in 1948 (after wartime service on radar in the RAF), and joined the group with Kendrew as my PhD supervisor. Francis Crick, also a physicist, with a particularly clear and powerful mind, and with distinguished wartime service at the Admiralty, joined in 1949. He, like Kendrew, had already embarked on a PhD at the beginning of the Second World War, and now became Perutz’s research student. Crick and I had both decided we wanted to apply ourselves in the biological field, and thought the MRC group was a good place to do so. It seemed obvious that the growing power of physical techniques should be able to reveal interesting and important new facts about biomolecular structures and mechanisms, just as had been the case in the physical sciences over the previous 50 years. I suppose that Max may have been influenced in his decision to take on his three colleagues by their histories of successful work, even if in rather different spheres. They in turn may have been more willing to enter an uncharted field for the same reasons. Sheer luck also played a part in having a good mixture of talents and personalities at hand.

It all provided a very pleasant and stimulating environment, particularly as we all lived in relatively close proximity, and in Cambridge. I was given complete freedom to choose my own problem, and after a little experience with crystalline proteins, I began low-angle X-ray diffraction work on live muscle in 1950, which soon provided promising new clues to the contraction mechanism. Crick also began work on crystalline proteins, but was joined in 1951 by Jim Watson (strongly recommended to Kendrew during a US visit), and diverted into DNA structure. I went to MIT as a postdoc in 1952, to learn how to apply the new art of electron microscopy to muscle structure, for I had realised what a powerful and complementary combination it would provide to X-ray results.

1953 was the miraculous year when everything first came together. Francis and Jim produced their double helical model of DNA, which promised, in principle, how to explain heredity and replication. Max showed that, contrary to previous expectations, the heavy atom phasing technique would indeed work on protein crystals. John began organising the equipment, assistants and computing to do this at high resolution, a major undertaking. At MIT, I was able to get electron micrographs of muscle cross-sections which confirmed directly my X-ray prediction of a double hexagonal array of separate filaments of actin and myosin, and, with Jean Hanson, was able to put forward the overlapping filament model for striated muscle structure, and to suggest the sliding filament mechanism, from other X-ray results.

Francis was joined in 1957 by Sydney Brenner, a very brilliant South African who had been working on phage and bacteria in Oxford, and they began all their very successful work in establishing the non-overlapping triplet code, and many other features of the translation machinery of DNA via RNA into protein. John was able to publish the first low resolution (6Å) three-dimensional myoglobin structure in 1958, showing the complicated folding of the polypeptide chain and the variously oriented α-helical parts of the structure. A higher-resolution structure (2Å) was published in 1961, in which individual amino-acid residues could be identified. In 1960, Max published a 5.5Å structure of haemoglobin, showing four exactly myoglobin-like chains packed together in one molecule, and proving beyond any doubt that these were indeed true structures. So now, in principle, the structure of any protein that could be crystallised in isomorphous form, with a number of different heavy atoms attached, could be determined to atomic resolution, and its functioning made accessible to detailed study and understanding.

By this time I had been working in Bernard Katz’s biophysics department at University College London for several years, where the MRC had provided me with a new Siemens electron microscope. I’d been able to get ultra-thin (~100Å) longitudinal sections of striated muscle which showed single layers of actin and myosin filaments with cross-bridges between them, and helped to convince more people of the validity of our model. I had also developed a negative staining technique, with phosphotungstic acid, and demonstrated the central channel in tobacco mosaic virus, in 1956. Later, with Geoffrey Zubay, I had worked on a spherical virus (Turnip Yellow Mosaic), suggested and supplied by Aaron Klug, another very clever migrant from South Africa, who was then at Birkbeck College working with Rosalind Franklin’s group, in Bernal’s department. He was studying this virus by X-ray diffraction, and we obtained good EM evidence for the 532 symmetry which his results indicated. This was the beginning of all the work on three-dimensional image reconstruction from electron microscope images which Aaron, John Finch and their colleagues and visitors did later in Cambridge. Brenner and Horne had improved the negative staining technique in 1959, and I made the further improvements of using uranyl acetate and holey carbon films, and demonstrated the structural polarity of actin and myosin filaments from muscle.

These various successes in Cambridge and in London provided a clear case for much further expansion of the laboratory in Cambridge, and for providing more room for visiting workers, who wanted to learn about the new discoveries and who would make it possible to explore them much further. Insufficient space was available in any of the University departments in the centre of Cambridge, and eventually the MRC (now headed by Sir Harold Himsworth, another very effective champion of the group) was able to find a suitable site for a relatively large new lab on the new Addenbrooke’s Hospital site, a mile or two out of town, but still within easy cycling distance down Hills Road. Fred Sanger, who had already won one Nobel Prize, but who was working under cramped conditions in the biochemistry department, had also expressed his willingness to join the new lab. His work on sequencing RNA and DNA was, of course, a splendid counterpart to the studies in Francis and Sydney’s group. I wanted to do further X-ray diffraction work on muscle, so I welcomed the opportunity to rejoin the group in Cambridge when the new lab opened in February 1962. Aaron Klug moved there at the same time with the rest of his group – Ken Holmes, John Finch, Bill Longley and Reuben Leberman – all sharing the same interests and hopes about biological structures.

Even before the new lab opened, several visiting scientists had already arrived, including those involved in working out the first two protein structures. As the work of the lab became better known, the number of visitors grew, no doubt helped by the timely award of Nobel Prizes later in 1962 to Max and John (Chemistry) and to Francis and Jim (Physiology or Medicine), shared with Maurice Wilkins at King’s College, London. Formally, the lab was run by a board, consisting initially of all the scientific staff who were already Fellows of the Royal Society, with Max as chairman. But in Max’s own words: ‘The Board never directed the laboratory research, but tried to attract or to keep talented young people, and give them a free hand. My job was to take an interest in their research, and to make sure they had the means to carry it out.’

In many ways, the lab was a kind of paradise for dedicated research scientists. No one had to do any teaching, or to write grant applications, or even to publish frequently. Expert practitioners in most of the relevant fields were readily at hand. Within reason, all supplies and equipment were either immediately available or rapidly obtainable, via Michael Fuller. The lab had sizeable mechanical and electronic workshops, where any novel equipment that the experimental groups needed and designed could be skillfully constructed and maintained. There were also good facilities for do-it-yourself construction work. Such arrangements followed the traditions of the Cavendish Laboratory. There was a democratically organised cafeteria, where everyone went for morning coffee, lunch and tea, and much scientific conversation and advice was always available. This was of great importance. People worked extremely hard and collaborated with each other extensively, because of the excitement and satisfaction of getting important scientific experiments to work properly, and to find out more and more fascinating new facts about nature.

I therefore thought it would be interesting and valuable, even at this late stage, to bring together some first-hand accounts, from the visitors’ points of view, of this particular scene of the origins of molecular biology. So I sent the following letter to a representative assortment of visiting scientists who had worked with the MRC group during the years 1957–86, which covered the period of the greatest initial activity:

June 2012

Dear Colleague,

As you may know, this year is the 50th anniversary of the opening of the LMB in Cambridge, England, and next year the new Laboratory will be opening there. John Finch’s volume gave a good history of the Lab from an internal standpoint, but I have long thought that a very important part of scientific history lies in the experiences of the visiting workers, particularly those from the United States, in the earlier years of the Lab, when it played such an important role in helping to establish the groundwork of molecular biology.

What I had in mind was a collection of essays, up to 3,500 words or so, by people like yourself, describing briefly the scientific problems in your own field of interest when you came to work in the Lab; how the scientific environment there influenced your work and your ideas about how a lab should best function (if it indeed did!); and what the subsequent developments in the field were that led to your own great scientific successes. You could also comment on the differences in style and personalities, in the Lab and outside it, between the USA and the UK at that time, and any personal reminiscences of Cambridge then.

Would you be interested in doing this, in the next four months or so – say by the end of October? It would make very interesting reading, and would preserve important information that otherwise will be lost.

With best wishes

Hugh Huxley

(former Joint Head of the Structural Studies Division, and Deputy Director of the Laboratory)

A little while later I sent a second short letter making a few suggestions about the style of the contributions:

Dear Colleagues,

I thought I should write a few lines to let you know how the plan to gather together a representative collection of memoirs of LMB alumni has been developing. Basically, extremely well, with about 45 acceptances, usually with great enthusiasm, and in fact representing a high proportion of those approached.

One thing I wanted to stress was the desirability of setting the detailed description of the particular line or area of work that you have followed in a somewhat broader framework, so that it would not only be interesting to the cognoscenti (and also provide an important historical record of what actually happened), but would also be as comprehensible as possible to those with a more general background, but interested in how molecular biology developed during those years.

Not easy, but please try!

I look forward to receiving your contributions by the end of October.

Best regards

Hugh Huxley

In the end, I received 41 contributions, from about 60 people contacted, with the missing ones usually caused by lack of time. There was considerable variation in the individual lengths, but the average was about 3,140 words. In the present volume, these memoirs are arranged in chronological order. The first two have, in fact, been published before, but they are of such interest in describing how the first two protein structures were solved that I thought them very worthy of inclusion. Also included is a particularly interesting piece by Sidney Altman, also published previously.

I hope that this volume will provide at least a small record of great days.

Figure 0.1: Hugh Huxley, first skiing holiday, New Year 1949, near Kuhtai, Austria (on Max’s recommendation).

CHAPTER 1

Myoglobin: A Whale of a Structure! (1957–9)

Richard E. Dickerson

Molecular Biology Institute, University of California, Los Angeles, CA

INTRODUCTION

How is a crystal put together? The picture in many people’s minds is of atoms or ions packed tightly together like tennis balls in a large cardboard box. Some crystals have only one type of ball, such as carbon atoms in diamond. Other crystals have two different kinds, such as sodium and chlorine ions in NaCl. But crystalline proteins or crystalline DNA? Ridiculous!

In 1953, I joined the laboratory of Bill Lipscomb at the University of Minnesota to work on crystal structures as a step towards a doctorate in physical chemistry. When I arrived, on the ‘Colonel’s’ (Lipscomb was from Kentucky) desk was a weird skeleton of linked triangles forming a fragment of an icosahedron, with one half open like a football helmet. This was the structure of a boron hydride, the Colonel told me, with boron atoms at the corners of the triangles and hydrogen atoms attached in unexpected places. I remember being astounded that such an awkward and ungainly entity could ever be induced to form a crystal. Little did I realise that in Cambridge, England, two colleagues named Perutz and Kendrew were, at that very moment, well on their way towards solving the crystal structures of molecules of vastly greater complexity than mere boron hydrides.

Things were much simpler in those distant days. I earned my doctorate with the aid of 375 X-ray reflections by solving the crystal structure of B9H15.1–3 The project had been begun by a visiting professor from Leeds University, Peter Wheatley, and I inherited it when he returned to England. Data were collected on film with a precession camera, spot intensity was gauged by visual comparison with a standard scale, and calculations were carried out on a desk calculator with the help of an IBM 407 punched-card machine, which could perform simple arithmetic operations on data entered via punched cards. IBM 407 was ‘programmed’ by connecting wires on a plugboard, rather like a primitive telephone switchboard. For the production of electron density maps, punched-card data were sent to Ray Pepinsky at Penn State University, who had developed the X-Ray Analogue Computer or X-RAC specifically for crystal structure analysis.3

Things improved vastly when the Colonel persuaded Honeywell Corporation in nearby St Paul to let us use their Remington Rand UNIVAC 1103A computer after office hours. The University of Minnesota had no digital computers of any kind at that time. They had considered purchasing an IBM 650 magnetic drum computer but decided that there was too little demand for its services to justify spending $200,000–400,000 on such a machine. The UNIVAC 1103A was a ‘huge’ computer, with 1,024 36-bit words of electronic memory and 16,384 words on a magnetic drum. Compare that to 2 GB or 2,000,000,000 bytes of storage on a removable flash drive memory stick for a modern laptop computer. One byte in modern usage corresponds roughly to 8 bits, so today’s 2 GB laptop memory stick has more storage capacity than 25,000 UNIVAC 1103A computers! As I was finishing my degree, a young postdoctoral fellow from Glasgow, Michael Rossmann, showed up on the scene. He and Audrey, and my wife Lola and I became good friends, but when I left Minneapolis in February 1957 I never dreamed that, in only a couple of years, we would be working together again, a third of the way around the world.

I did not go straight to Cambridge. In fact, the Colonel told me that Oxford and Cambridge were old hat, and that I would be better off at Leeds University, which offered such groundbreaking crystallographers as Peter Wheatley, Durward Cruickshank and George Jeffrey. So one cold day in February 1957, Lola and I disembarked at Southampton and took a train north to Leeds, where Peter Wheatley met us at the station. But things were not what they might have been. Jeffrey had finally moved permanently to the University of Pittsburgh where, over the years, he built up a large and productive Graduate Crystallography Department, the only one of its kind in the United States. Durward Cruickshank had moved to Manchester, which had the digital computer (the Ferranti Mark I) that Leeds University lacked. (When Leeds obtained its first computer a few years later, the university housed it in a former Methodist church building adjacent to the campus. In a bizarre fit of inspiration, its official name was the ‘Leeds University Computer and Integrator, Ferranti’ or LUCIFER.)

At Leeds, I collected precession data on a now mercifully forgotten dimethylsulfoximine, had a Patterson function calculated on the Manchester computer, and then solved the three projections of the structure using Beevers–Lipson strips and a desk calculator. But during the summer of 1957, a crisis arose. As I have related before,4,5 Wheatley announced that he could no longer support a wife and two children on his annual academic salary of £900 and was leaving Leeds to head up a new Monsanto crystallographic laboratory in Zurich.

By a stroke of extraordinarily good fortune, at that very moment, Max Perutz and John Kendrew in Cambridge were advertising for one or more postdoctoral fellows to work with them on the high-resolution crystal structure of myoglobin and the low-resolution structure of the larger haemoglobin. Perhaps in a fit of conscience, Bill Lipscomb proposed my name and provided a very positive recommendation. Peter Wheatley also suggested me, and the result was that I received a phone call from Max, inviting me down to spend a day talking and looking around the laboratory. During the day, we all visited the Royal Institution in London to talk with David Phillips, who was collaborating with the Cambridge team. It was there that I first met a wonderful person, Sir Lawrence Bragg, the previous director of the Cavendish Laboratory at Cambridge and now the director of the Royal Institution. It was Bragg who, together with Max Perutz, had built the Medical Research Council (MRC) Laboratory in Cambridge and attracted to it people such as John Kendrew, Francis Crick and Jim Watson. In 1915, the 25-year-old Bragg had shared the Nobel Prize in Physics with his father, W.H. Bragg, for essentially inventing X-ray crystal structure analysis. I looked at the man who had created Bragg’s Law and suppressed an urge to exclaim: ‘I assumed you were dead!’

There was a meeting of the minds, and I accepted their invitation to join the MRC group at Cambridge in the autumn of 1957. I chose to work with John Kendrew in part because the idea of getting a structure at atomic or near-atomic resolution appealed to a small-molecule crystallographer.

GETTING STARTED

The MRC Laboratory of Molecular Biology occupied a long, one-storey temporary building that had been built as a metallurgical laboratory during the Second World War. It was known irreverently as ‘The Hut’ and sat in a courtyard of the Cavendish physics laboratory. The Hut contained a wet laboratory and offices, but the X-ray machines themselves were mercifully housed in the basement of the adjacent and more sturdily constructed Cavendish laboratory. The wet laboratory was at one end of the Hut. One side of the central corridor had offices for Max Perutz, Francis Crick and Sydney Brenner. On the other side were the offices for John Kendrew and for postdoctoral fellows. When I arrived in the autumn of 1957, I shared this office with Erwin Alver and Roger Hart. Alver, from the University of Oslo, was an inorganic crystallographer like myself. Roger Hart was somewhat more qualified: although originally from the United States, he had first worked with Rosalind Franklin, and then with Aaron Klug after her death.

Alver only stayed for one year and returned to Oslo in the summer of 1958, shortly after the arrival of a third colleague, Bror Strandberg, from Uppsala. Alver worked with Kendrew like myself, and Hart worked with Perutz. Prior to Bror’s arrival, Alver and I joked that we were the only two people in the MRC laboratory who had ever completely solved a crystal structure, although mine had only nine boron atoms and Alver’s was comparably insignificant. Proteins constituted an entirely new universe for both of us.

Kendrew had initially studied horse myoglobin, but soon reasoned correctly that the oxygen-storing molecule in tissues should be especially plentiful in aquatic mammals that dived and remained under water for appreciable time periods. Finback whale and sperm whale myoglobins were isolated and tried, and the latter was selected because its myoglobin concentration was so high that the meat was almost black.6 Sperm whale meat was shipped from Peru and stored frozen in large garbage cans in a deep-freeze room of the Molteno Institute, down the road from the MRC laboratory. Myoglobin was isolated and purified by laboratory aides, and crystals were grown in 2 or 4 ml screw-top vials. Crystals grew so large that they had to be cut down to size with a scalpel blade before being inserted and sealed in glass capillaries for X-ray photography.

Roger Hart was unexpectedly an important source of inspiration to me in these early days. John Kendrew carefully demonstrated to me every step of the crystal-mounting process: choosing a good crystal, cutting it down to size, inserting it into the capillary and drying away excess solvent from around it, adding short plugs of solvent to both ends of the capillary to maintain a moist atmosphere, sealing both ends of the capillary with wax, and mounting it on a goniometer head for placement on a precession camera. I found this procedure incredibly difficult and became rather intimidated by the entire process. I began to wonder whether I would ever master the trick. But Roger gave me pause. His hands were twisted and crippled by polio, yet he was taking survey picture after survey picture of derivatives of haemoglobin for Max Perutz. I said to myself, ‘By God, if Roger can mount crystals with those hands, I most certainly should be able to.’ And so it was, after hours of trial and error. Only later did I learn that Roger did not actually mount the crystals that he was photographing. Perutz came in after hours and mounted the next day’s supply of haemoglobin crystals!

Bror Strandberg arrived from Uppsala in June 1958, and the project really got going. The third member of our myoglobin team was David Phillips in London. He was to collect data for one of the heavy-atom derivatives at the Royal Institution. John’s original plan had been to follow his 6Å low-resolution analysis with a higher-resolution analysis at 2.5Å. The 6Å analysis required about 400 independent X-ray reflections from parent myoglobin crystals and from each of the five heavy-atom derivatives. Increasing the resolution from 6Å to 2.5Å would necessitate collection of (6/2.5)3 × 400 = 5,500 reflections from each crystal type. But in planning discussions, someone pointed out that, at 2.5Å, we were not using the full power of the precession camera. The greatest resolution that we could obtain on our 5"×5" photographic plates was 2.0Å, for which the reflections would extend all the way out to the edges of the plate. Why not go for broke and collect 2.0Å data instead? We decided to do so and to collect and measure circa (6/2)3 × 400 = 10,800 reflections per data set. This would give us twice the information of the originally planned 2.5Å analysis. (In the end, after allowing for space group extinct reflections and other problems, a dependable set of 9,600 reflections was used in the 2Å analysis.)

Data were collected from four heavy-atom derivatives: para-chloromercuribenzene sulphonate, mercury diamine (HgAm2), gold chloride, and the double derivative para-chloromercuribenzene sulphonate/mercury. Each data set required 22 different precession camera photographs; for each photograph, a newly mounted myoglobin crystal was used to minimise radiation damage. In precession cameras, X-ray reflections or spots are arranged in a series of parallel lines. This meant that one could use a Joyce–Loebl microdensitomer to scan along each of these lines, yielding peaks that could then be measured with a millimeter scale. Max and John had a crew of technicians who were available to carry out this process, and we ended with a long list of (h,k,l) reflection indices and their corresponding peak intensities. These were then typed on punched paper tape for input to the Electronic Delay Storage Automatic Calculator (EDSAC) II computer in the nearby Cambridge Mathematical Laboratory. EDSAC II was considered a major step up from the EDSAC I that Kendrew had used for low-resolution myoglobin. Kendrew and his colleague Bennett in 1952 were actually the first people to program a digital computer or ‘digital electronic calculating machine’, the EDSAC I, to carry out a three-dimensional Fourier synthesis.7 Bennett subsequently moved to the Ferranti Corporation in Manchester, the place that also lured Durward Cruikshank away from Leeds. This new EDSAC II computer had 2,000 words of high-speed core storage and two magnetic tape drives. The core was far too small to permit shuffling and reordering of 10,000 X-ray reflections from each of five sets of data: a parent protein and four derivatives. Merging of data had to be accomplished by thumb-tacking strips of punched paper tape containing intensity data to a bulletin board, shuffling the strips manually to merge the data sets, and then running everything back into the computer and emitting one grand master tape. Figure 2.7 of Michael Rossmann’s chapter shows Bror and me walking our data bulletin board back from the computing centre. A good strong wind might have made a major change in our analysis.

Input to EDSAC II was performed by punched paper tape using standard British telegraph readers. Little metal fingers in the reader pushed up through the holes in the tape, reading the information in the pattern of holes. If an input tape was mispunched during preparation, it was ruined and could only be corrected by making a new tape. In desperation, the custom of patching the occasional mispunched hole with Scotch tape was developed. This worked fine unless the tape reader fingers pushed off the patch, causing a misreading and sometimes a tape jam. The computing centre came down on this with a hammer, threatening that any tape found to be patched in this manner would be confiscated. (I never recall this threat actually being carried out.)

The heart of EDSAC II was an array of long racks of vacuum tubes, with each rack being capable of being pulled out individually by its handle when one of its tubes blew. Wiring diagrams for the computer were in pencil in a large stack of circuit diagrams. When a change was made, the diagrams were erased and corrected. Whether permanent or even inked drawings of the computer existed or not, I was never aware of them.

There were three grades of access to EDSAC II: users, partially authorised users, and fully authorised users. Users could only have normal access to the computer during the day while it was manned by computer centre staff. Partially authorised users were allowed to run the computer as late as they chose and to turn it off when finished by pulling a set of wall switches in a defined order. Fully authorised users were permitted to turn the computer on and off. I eventually qualified as a partially authorised user. I do not know if any member of the protein crystal structure group ever rose to the rank of a fully authorised user, but if so, it was probably Michael Rossmann. His programs for difference Patterson and difference Fourier refinements were so complex that they were sometimes used for the morning computer check, instead of the standard system-checking routines.

I DON’T RECALL THE NAME, BUT THE PHASE IS FAMILIAR

We have not yet considered why five different sets of data, each of roughly 10,000 reflections, were necessary. This brings us to the phase problem, which is the key to the structure analysis of macromolecules. One can generate an electron density map of the protein by a process of Fourier synthesis. Each of the thousands of reflections in the X-ray pattern contributes one wave to the image of the protein. The direction of each wave is determined by the position of its reflection in the X-ray pattern, and the wavelength of the wave is inversely proportional to the distance of the reflection from the centre of the X-ray pattern. The amplitude of each wave is the square root of the measured intensity of the X-ray reflection.

So far, so good. This is nothing that a computer cannot handle. But one more – and very serious – complication remains. Each one of these thousands of waves has a phase – the fraction of a 360° cycle by which it is shifted compared to the other waves before they are all added together. To calculate the image of a protein, one must know both the amplitude and the phase of each of the 10,000 reflections. How does one find out the phase of each and every reflection in the X-ray pattern?

Each wave corresponding to one X-ray reflection is actually the sum of waves from all of the atoms in the scattering molecule. This sum, again, is a complex number. The amplitude of scattering from each atom depends on the number of electrons in the atom, and its phase depends on the position of the atom in the molecule. Figure 1.1a represents scattering from a single atom for one particular X-ray reflection. The length of the vector is the amplitude of the wave, and the angle of the vector from the horizontal or real axis is its phase. Figure 1.1b shows how scattering vectors from three atoms with similar numbers of electrons but different locations add together to produce an amplitude and a phase for the three-atom molecule as a whole.

One classic phase-solving approach for small molecules is the heavy-atom method. Tag the molecule with a heavy atom having so many electrons that it dominates scattering, as in Figure 1.1c. First, locate the heavy atoms in the unit cell. (See Michael Rossmann’s Chapter 2 for more on this issue.) Compute an electron density map using the phases of the heavy atom itself, which you can calculate if you know where the heavy atoms are located in the cell. If you are lucky, this map will show the heavy atoms plus at least a partial image of the rest of the molecule. Add more of these atoms as they become visible in the maps, and iterate until you have the picture of the complete molecule.

Figure 1.1. For a given X-ray reflection, addition of waves from different atoms in one molecule. (a) A single atom X. (b) A three-atom X3 molecule, with similar atoms in different locations. The heavy arrow represents scattering for that reflection from the entire molecule. (c) Result of tagging the three-atom X3 molecule with a bromine atom. Because the heavy atom has so many electrons, it dominates scattering, and its phase is very similar to that for the entire X3Br molecule.5

But it was not at all clear whether this approach would work with proteins. Haemoglobin, with a molecular weight of 68,000, would have something on the order of 34,000 electrons around its atoms. How could one imagine that one or two mercury atoms of 80 electrons each could dominate the phasing? Indeed, could one expect to see any change in X-ray intensities at all when 80 or 160 electrons are added to a molecule that already contains 34,000? In 1954, Max Perutz provided the Rosetta Stone for protein crystal structure analysis when he demonstrated that, although heavy atoms did not dominate phases from a protein, they did at least alter the intensities of the reflections in a manner that might allow one to deduce the phase angles of reflections from the parent protein.8 Classical heavy-atom phasing methods would not work, but isomorphous replacement phase analysis might.

The man who provided the methodology for this kind of phase analysis was David Harker, who in 1956 was trying to solve the structure of the protein ribonuclease at the Brooklyn Polytechnic Institute.9 Figure 1.2, reproduced from his work, shows how the scattering vector from the protein plus heavy atom (FO+X in his notation) is the vector sum of scattering from the protein alone FO and the heavy atom alone FX. (Throughout the present chapter, bold type represents vectors in phase space, and regular type is used for vector amplitudes). The Harker phase-circle diagram in Figure 1.3 for two heavy-atom derivatives, X and M, is produced by:

(1) Drawing a circle of radius FO around the origin

(2) Drawing a circle of radius FO+X around a point located at −FX

(3) Drawing a circle of radius FO+M around a point located at −FM.

FO, FO+X and FO+M are the measured amplitudes of X-ray scattering for that reflection from the parent protein and the two heavy-atom derivatives. FX and FM are the scattering vectors for heavy atoms X and M alone, which you can calculate, in both phase and amplitude, once you have located the positions of the heavy atoms in the unit cell. The location on the FO circle where all three circles intersect marks the correct phase angle for the parent protein.

Figure 1.2. For one X-ray reflection, addition of scattering vectors from a parent molecule FO and an added heavy atom FX to yield the observed experimental scattering from the heavy-atom derivative FO+X.9 Reprinted with permission from the International Union of Crystallography (http://journals.iucr.org).

Figure 1.3. Phase-circle diagram for one X-ray reflection, using experimental amplitudes from the parent molecule O and the two heavy-atom derivatives X and M. Each derivative phase circle crosses the parent circle O at two places. The intersection that is common to the two derivatives is the correct phase angle.9 Reprinted with permission from the International Union of Crystallography (http://journals.iucr.org).

Of course, nothing is ever as simple as it first appears. Errors in intensity measurements produce errors in the three phase-circle radii, which in turn can make phase-angle determination inexact. The solution is to collect data from three, four or five heavy-atom derivatives instead of the minimal two, and to draw the best phase angle through their cloud of intersections. Because myoglobin is so much smaller than haemoglobin, it was actually the first protein structure to be solved in three dimensions by Harker’s isomorphous replacement method.10,11 In the Kendrew Archives at the Bodleian Library in Oxford, there resides a three-ring notebook in which John Kendrew drew a set of four hundred 6Å Harker phase-circle diagrams with a ruler and a compass, using different-coloured pencils for each derivative. When we began preparing for 2Å high-resolution phase analysis in 1958, John handed me this notebook as a guide to what I should incorporate into a computer program. Today, the notebook sits jealously guarded in the Kendrew Archives at the Bodleian, and the photocopies that I obtained recently are not of publication quality. I wish that I had had access to a really good colour photocopy machine half a century ago. But Figure 1.4, from the published work, will have to do.

Figure 1.4. Phase circles for three X-ray reflections from myoglobin at 6Å resolution, using parent myoglobin and five different heavy-atom derivatives. (a) An excellent, unambiguous phase determination. (b) A somewhat less well-defined phase. (c) A poorly defined phase angle.11

The most serious problem with phase analysis at 2Å was how to obtain information from poor phase determinations such as Figure 1.4c. Several approaches were tried.12,13 Michael Rossmann proposed and Hilary Muirhead programmed a ‘least scatter’ approach, choosing as a phase the radial line along which intersections with phase circles lay most closely together. Roger Hart used a function to calculate the probability of any particular phase angle, and then chose the direction that led to maximum probability. But the optimal solution was developed by Blow and Crick.14 Using lack-of-closure errors as defined in Figure 1.5, they calculated the phase-angle probability all around the 360° phase circle (Figure 1.6), and then chose the centroid or centre of gravity of that probability distribution as the ‘best’ phase angle. The vector to this centroid, m in Figure 1.6, then yields the best phase φB for that reflection in the Fourier synthesis and the optimal weighting factor m. The length of that vector m is 1 for a perfect phase determination and 0 for a hopelessly bad determination. The length m itself is then a measure of the dependability of that particular phase angle and is called the ‘figure of merit’.12,13 The mean figure of merit provided a way of assessing the quality of the entire phase analysis.

Figure 1.5. Lack-of-closure errors in phase analysis. In this diagram, the amplitudes of the protein and its two heavy-atom derivatives are represented by F, F1, and F2. Scattering from heavy atoms alone is shown as vectors f1 and f2. For derivative 1, the requirement that F1 = F + f1 is satisfied at points A and B around the parent phase circle; for derivative 2, the requirement F2 = F + f2 is met at points B and C. Hence, the true phase angle is found at the shared point B. But for the general phase angle φ, the measured F1 from derivative 1 is too long by distance ε1, and that for derivative 2 is too short by distance ε2. These lack-of-closure errors can be used to calculate the probability of correctness of any angle. At the correct point B, this probability rises, in principle, to unity.12 Reprinted with permission from the International Union of Crystallography (http://journals.iucr.org).

Figure 1.6. Phase-angle probabilities plotted around a 360° phase circle of unit radius. This particular example exhibits two Gaussian-like peaks of probability, one roughly twice the other. Vector m marks the centre of gravity of the probability curve.12 Reprinted with permission from the International Union of Crystallography (http://journals.iucr.org).

I wrote a program for EDSAC II that calculated phase probabilities and defined the centroid vector. Figure 1.7 exhibits three examples from the high-resolution myoglobin analysis and shows the usefulness of the figure of merit m as a measure of the quality of each phase determination. Calculation of phase angles was a slow process by modern standards:

With four heavy-atom compounds, the time required for one phase determination on EDSAC II was 3.5 sec, of which about 1 sec was occupied in punching results. The full set of 9,600 reflections required 10 hr.12

The mean figure of merit m ranged from 0.90 at the centre of the X-ray pattern (lowest resolution) to 0.45 at the high-resolution 2Å limit. We experimented with various exponential sharpening functions. One approach was simply to accept the figure-of-merit results, with its falloff of amplitudes in the high-resolution region of the diffraction pattern. But this damped down the calculated image too much and smeared out fine structure features. The best solution was to apply a sharpening function so that the average falloff of the ‘best’ data, including figures of merit, matched the average falloff of the original experimental data set. This had the result of keeping the effective resolution the same, but emphasising well-established phases and damping down those that were badly underdetermined.

Figure 1.7. Examples of phase determination for three X-ray reflections in the 2Å phase analysis. The figures of merit are 0.96 (a), 0.59 (b), and 0.05 (c), which was the most poorly determined phase in the entire 2Å myoglobin analysis. Position m in each diagram marks the most probable phase, and position b is the ‘best’ phase using the centroid of the full probability circle. In the single-peak function (a), the most probable and best phases are the same. In the two bimodal functions, the best phase is shifted away from m somewhat towards the secondary peak Y. Reprinted with permission from the International Union of Crystallography (http://journals.iucr.org).

THE ALL-NIGHT PARTY

For the last stages of myoglobin analysis – phase refinement, electron density calculation and interpretation of results – myoglobin had a three-man team. David Davies from the National Institutes of Health in Washington, DC, joined us in April or May 1959 and, like Bror, remained for a time after I departed for the University of Illinois in September 1959. Once the weighted phase angles had been calculated, the next step was to calculate the 2Å electron density map of myoglobin and see what new information could be learned. The main question was whether those sausage-like rods seen in the 6Å map actually were α-helices. There was a strong presumption of this at 6Å, but no proof. Moreover, the connections between ‘sausages’ in the 6Å map were unclear in places, especially where they appeared to navigate a sharp corner.

Calculation of the three-dimensional electron density map of myoglobin, involving 9,600 reflections, was an all-night affair at the EDSAC II computer. John Kendrew, Bror Strandberg, David Davies and I, as well as our London collaborator David Phillips and some of his coworkers, were all on hand. Ninety-six sections were calculated through the crystals, with each section being a grid of density values spaced roughly 2/3Å apart.15 The complete electron density map required twelve hours of computer time. In contrast, calculation of the earlier 6Å map with 400 X-ray reflections had taken only 72 minutes on EDSAC I.11

As the night dragged on and as completed electron density sections began appearing, the sections were scanned for information that the low-resolution map did not have. The haem group was easily identified (Figures 1.8 and 1.9) and, to everyone’s satisfaction, the 6Å ‘sausages’ did indeed turn out to be α-helices. Side chains were clearly visible, and even C=O groups within the helix were clear, thereby establishing the direction of the backbone chain (Figure 1.10). The overall structure of the sperm whale myoglobin molecule is shown in Figure 1.11.

To celebrate the new structure, a garden party was held at dusk on the lawn of Peterhouse. Contoured Plexiglas sheets of the 2Å myoglobin map were stacked on a large light box that was the centre of attraction. One of my fondest memories of that party is that of Sir Lawrence Bragg, who had pushed for protein structure analysis for more than a quarter of a century, dragging one party attendee after another over to the light box, pointing down the E-helix, and saying in exultation, ‘Look! It’s hollow! It’s hollow!’

Figure 1.8. Section through the electron density of the haem group in the 2Å structure analysis, with an ideal framework superimposed. Positions of the ‘ears’ extending from the four five-membered rings are clearly established.15

Figure 1.9. View through stacked Plexiglas sheets showing the haem group seen on edge and a view down the hollow F-helix that is attached to the haem by a His side chain. Map, above; interpretation, below.15

Shortly thereafter, my wife Lola, my infant son Ian (a Cambridge citizen by birth), and I dashed down to Southampton to catch a ship home so I could take up my duties as a new faculty member at the University of Illinois. Bror and David Davies remained some months longer, helping John construct a display of electron density built of coloured clips on a forest of steel rods (Figure 1.12), into which one could build an accurate atomic structure using wire models invented by John Kendrew. (Kendrew model parts are now a collector’s item.) The model was built in the basement of the Cavendish Laboratory and was put on proud display the following autumn, when the International Union of Crystallography held its biennial congress in Cambridge. For the congress, the new myoglobin and haemoglobin results were displayed in a room that belonged to the Electrical Engineering Department. Permission for use of the room was granted, but John and Max were told in no uncertain terms to get ‘all of that stuff’ out of the room quickly before the autumn term began. The models now reside in the Science Museum in Kensington, London.

Figure 1.10. Density in an unrolled α-helix at 2Å. Not only are side chains clearly visible above and below the plane of this unrolled cylinder, the C=O groups along the backbone are so well defined that they establish the direction in which the α-chain runs. The 6Å ‘sausages’ are clearly Pauling/Corey α-helices.15

Figure 1.11. An accurate but simplified diagram of the folding of α-helices in sperm whale myoglobin from the 2Å analysis. Each black circle along a helix marks the α-carbon of an amino acid. Zigzag lines follow the chain backbone in nonhelical regions such as bends between helices. The haem group and its attachments are also shown. Drawn by the author to advertise a 1962 lecture by John Kendrew at the University of Illinois.5

Figure 1.12. The 2Å electron density displayed as coloured clips in a forest of steel rods mounted on a plywood base. The detailed backbone of the polypeptide chain can be seen snaking through this forest. John Kendrew (centre left) explains myoglobin features to attendees at the 1960 meeting of the International Union of Crystallographers in Cambridge.5

Not long after the low-resolution 6Å myoglobin structure was published, someone in Howard Schachman’s laboratory at Berkeley drew the cartoon seen in Figure 1.13. John Kendrew stands uncertainly to the right of the monster that he has created, with computer output strewn on the floor. Schachman sent the drawing to me in 1965, but could not recall just which of his students had been the artist. The molecule clearly has the same breaks between helices and the superfluous connections that were relics of the low-resolution structure (reference 10).

Figure 1.13. Dr Frankendrew’s monster. The 6Å model, caricatured around 1958 by a now-forgotten member of Howard Schachman’s laboratory at the University of California, Berkeley. The caricature was sent to me by Schachman in 1965. Note the breaks and false connections between helices that were cleared up by the 2Å analysis.5

AFTERTHOUGHTS

John Kendrew and Max Perutz had quite different styles in running a research laboratory. John was the mentor, guide and organiser. He was simultaneously involved in several other activities outside the laboratory: renovation of the art in Peterhouse and renovation of the electrical system that (as I remember) Rutherford had first installed, science advisor to the British government, liaison with the US government over the Polaris missile programme, and other useful activities. He would come into the laboratory for a group meeting several mornings per week and would ask us: ‘How is progress? What has been happening? What do you plan to do next? What do you need?’ He was valuable to the group as a resource and inspiration. In a sense, he was an excellent mentor for someone who aspired to run an independent research group some day. We learned how to run our own show. Myoglobin, however, marked the end of John’s scientific research career. In 1974, after the conclusion of the final 1.5Å-resolution analysis of myoglobin by Herman Watson and coworkers, John left Cambridge to become the first director of the European Molecular Biology Organization Laboratory in Heidelberg. By all accounts, he did a superb job. When he retired from there in 1981, he accepted a position, appropriately enough, as President, or Master, of St John’s College at Oxford. (This is why all his files eventually wound up at Oxford instead of Cambridge.) I can imagine some awe-stricken fresher coming up to the distinguished, white-haired president and asking timidly, ‘Are you the St John for whom the college is named?’ In contrast, Max was a hands-on bench biochemist whose centre of gravity was always the laboratory itself. On many occasions, he would involve himself directly in the ongoing structure analysis as, for example, when he came back to the laboratory in the evenings to mount crystals for Roger Hart’s next-day precession camera surveys. He became director of the MRC laboratory and served for many years, but this did not stop his daily involvement with the lab. Both styles had their merits: one learned from John, but one learned with Max. Max’s lifelong commitment to research is acknowledged in Figure 1.14, which depicts him as the modern Noah of the MRC, bringing proteins safely through the deluge on an ark that he himself constructed. The approaching dove at right carries the reward that Max and John earned for their efforts.

Figure 1.14. Max as Noah, bringing his proteins safely through the deluge. Drawn by editorial cartoonist Dave Granlund after my rough sketches for the cover of Present at the Flood: How Structural Molecular Biology Came About.5 Note, to be rigorously fair, that the myoglobin molecule leading off at the prow of the Ark more properly belongs to John Kendrew, not to Max. But it was indeed first. For more superb cartoons by Granlund, see his website: www.davegranlund.com/cartoons.

In 1996, the International Union of Crystallography held its congress in Seattle, WA. A panel of crystallographic Nobel laureates were invited to speak about their lives in structure analysis. One question asked by the moderator was: ‘If you had to live your life over again, would you do again just what you actually did?’ My graduate mentor Bill Lipscomb and most other Nobel laureates replied, ‘Of course.’ But John Kendrew had a different take on things: over the years, he had come to appreciate how much he enjoyed scientific administration at the European Molecular Biology Organization Laboratory. If he had to do it over, he said, he probably would not have spent so many years on research, but would have moved into administration much earlier. But there is an interesting flaw in this argument. He was presumably offered the European Molecular Biology Organization position because he was both a capable administrator and a Nobel laureate. Had he not solved the myoglobin structure or carried out some comparable notable scientific achievement, it was unlikely that he would have been chosen as the founding leader of the European Molecular Biology Organization Laboratory. But all things turned out for the best for both John and Max. De mortuis nil nisi bonum.

REFERENCES

1. R.E. Dickerson, P.J. Wheatley, P.A. Howell and W.N. Lipscomb, 1956. Boron arrangement in a B9 hydride. Journal of Chemical Physics 25, 606–07.

2. R.E. Dickerson, P.J. Wheatley, P.A. Howell and W.N. Lipscomb, 1957. Crystal and molecular structure of B9H15. Journal of Chemical Physics 27, 200–209.

3. R.E. Dickerson and W.N. Lipscomb, 1957. Semitopological approach to boron-hydride structures. Journal of Chemical Physics 27, 212–17.

4. R.E. Dickerson, 1992. A little ancient history. Protein Science 1, 182–6 (reproduced in reference 5).

5. R.E. Dickerson, 2005. Present at the Flood: How Structural Molecular Biology Came About. Sinauer Associates, Sunderland, MA.

6. J.C. Kendrew and R.G. Parrish, 1956. The crystal structure of myoglobin: III. Sperm-whale myoglobin. Proceedings of the Royal Society of London A 238, 305–24.

7. J.M. Bennett and J.C. Kendrew, 1952. The computation of Fourier syntheses with a digital electronic calculating machine. Acta Crystallographica 5, 109–116.

8. D.W. Green, M. Ingram and M.F. Perutz, 1954. The structure of haemoglobin: IV. Sign determination by the isomorphous replacement method. Proceedings of the Royal Society of London A 225, 287–307.

9. D. Harker, 1956. The determination of the phases of the structure factors of non-centrosymmetric crystals by the method of double isomorphous replacement. Acta Crystallographica 9, 1–9.

10. J.C. Kendrew, G. Bodo, H.M. Dintzis, R.G. Parrish, H. Wyckoff and D.C. Phillips, 1958. A three-dimensional model of the myoglobin molecule obtained by X-ray analysis. Nature 181, 662–6 (in reference 5).

11. G. Bodo, H.M. Dintzis, J.C. Kendrew and H.W. Wyckoff, 1959. The crystal structure of myoglobin: V. A low-resolution three-dimensional Fourier synthesis of sperm-whale myoglobin crystals. Proceedings of the Royal Society of London A 253, 70–102.

12. R.E. Dickerson, J.C. Kendrew and B.E. Strandberg, 1961. The crystal structure of myoglobin: phase determination to a resolution of 2 Å by the method of isomorphous replacement. Acta Crystallographica 14, 1188–95.

13. R.E. Dickerson, J.C. Kendrew and B.E. Strandberg, 1961. The phase problem and isomorphous replacement methods in protein structures. Computing Methods and the Phase Problem in X-ray Crystal Analysis pp. 236–51, Pergamon Press, New York.

14. D.M. Blow and F.H.C. Crick, 1959. The treatment of errors in the isomorphous replacement method. Acta Crystallographica 12, 794–802.

15. J.C. Kendrew, R.E. Dickerson, B.E. Strandberg, R.G. Hart, D.R. Davies, D. C. Phillips and V.C. Shore, 1960. Structure of myoglobin: a three-dimensional Fourier synthesis at 2 Å resolution. Nature 185, 422–27 (in reference 5).

FURTHER READING

R.E. Dickerson, 2005. Present at the Flood: How Structural Molecular Biology Came About (Sinauer Associates). This is a collection of reprints of key papers, along with explanation and discussion. Three of its chapters –

Chapter 5: The Race for the Double Helix

Chapter 6: How to Solve a Protein Structure

Chapter 7: High-Resolution Protein Structure Analysis

– specifically describe research initiated and carried out at the Medical Research Council Unit that became the MRC Laboratory for Molecular Biology.

CHAPTER 2

Recollection of the Events Leading to the Discovery of the Structure of Haemoglobin (1958–64)

Michael G. Rossmann

Department of Biological Science, Purdue University, West Lafayette, IN

INTRODUCTION

My PhD work in the laboratory of Monteath Robertson at the University of Glasgow in Scotland required lengthy calculations of Fourier series summations to determine electron densities. Our computational equipment consisted of desk calculators powered by electric motors and Beevers–Lipson strips representing sine and cosine curves with different amplitudes. It used to take me about one week to calculate one two-dimensional projection for the flat aromatic molecules I was analysing. It was, therefore, a great delight for me to encounter at the University of Minnesota in Minneapolis one of the first commercial computers, the UNIVAC 1103, when I joined the laboratory of Bill Lipscomb (the ‘Colonel’) as a young postdoctoral fellow. The Colonel had arranged for a few hours per week of computing time for his group on the UNIVAC computer at Honeywell in neighbouring St Paul. I overlapped with Dick Dickerson for a couple of months as he finished his doctorate on the structure of boron hydrides, a subject that gained the Nobel Prize for the Colonel about fifteen years later. Dick taught me my first lessons on how to program a computer. Before I left Minnesota two years later, Bob Jacobson, a graduate student with the Colonel, and I had written a fairly complete crystallographic computing package that continued to be in use for many years.

While in Minnesota, I attended the International Union of Crystallography meeting in Montreal and listened to a fascinating lecture by Dorothy Hodgkin1 on the structure of myoglobin being tackled by John Kendrew in Cambridge. Work on the structure determination of proteins seemed, to me, to be the best kind of project for my interests in methods for solving the crystallographic phase problem and the use of modern computational techniques. Fortunately Max Perutz, the director of the Cambridge laboratory that also included John Kendrew, quickly replied positively to my request for a job.

When my family and I arrived in Cambridge, I found a kind of environment different from what I had known in either Glasgow or Minneapolis. We worked in the little ‘Hut’ (still standing even today; Figure 2.1) in the New Museum Site off Free School Lane, close to the central market square. I gathered that there was insufficient space in the Cavendish Laboratory, which was world-famous for the work of Thomson, Rutherford and others on the structure of the atom, to also accommodate Max Perutz and members of his group who were studying biological molecules. Thus, we were all crowded into the Hut, next to the Austin Wing of the Cavendish. I shared a very small office with Bror Strandberg, a Swedish graduate student; Dick Dickerson, who had arrived via a circuitous route after a short stay at the University of Leeds; and Larry Steinrauf, a former student of Linus Pauling. The office opposite ours housed Francis Crick and Sydney Brenner. Max and John each had their own office. There was one office for the ‘computer girls’ who helped with manual calculations, another office for Ann Cullis (assistant to Max Perutz) and Mary Pinkerton (assistant to John Kendrew), an office for a graduate student (Hilary Muirhead) and assorted postdoctoral fellows who worked for Sydney and Francis, plus a very crowded biochemistry laboratory. Everyone packed tightly into the central corridor of the Hut at 11.00 each morning, reaching for the coffee pot brewed by Leslie Barnett, Sydney Brenner’s assistant, while debating everything from the relation between church and science in the then new Churchill College to the best ways of finding heavy-atom positions in protein crystals. Many a morning the conversations were dominated by Francis’s loud but appealing voice and provocative ideas. This was, for me, an intellectual environment more stimulating than I had ever previously encountered. I started to realise that biology was a more interesting subject than I had thought and to recognise how ignorant I had remained in my narrow undergraduate physics and mathematics education. No longer would the phase problem be my only challenge, as I became aware that the most significant problems were those of the way the universe and Earth had evolved to make the present day.

Figure 2.1. The Medical Research Council Hut at the New Museum Site off Free School Lane, Cambridge, photographed in 2003, still almost unchanged after nearly 50 years. However, in 2003, it was all locked up, whereas in 1959, it was full of activity day and night.

PROTEIN STRUCTURE IN CAMBRIDGE BEFORE 1958

The likelihood of ever solving the three-dimensional structure of a protein seemed extremely small even in the 1950s. There were too many data to be collected, no reasonable way of solving the phase problem, and no way of computing the results to make an interpretation of the collected data. But maybe even more daunting was the nagging question of whether each protein molecule had a unique structure. To some extent, these problems were starting to be solved. Bernal and Dorothy Crowfoot (later Hodgkin) had shown that pepsin could be induced to grow crystals that could diffract to near-atomic resolution,2 implying that all the molecules in the crystal had the same structure. Max encouraged Tony Broad to build a powerful rotating-anode X-ray generator. Max had also overseen the development of an accurate instrument for measuring the intensity differences between the X-ray reflections from isomorphous heavy-atom derivatives of haemoglobin crystals. Punched-card-driven devices, which were first used in the English textile industry and later became standard items of office equipment as the forerunners of computers, were starting to be used for mathematical computations. Furthermore, Max had been creating a set of very original techniques for solving the phase problem. One of these was the isomorphous replacement technique that had been the basis of the first crystal structures ever solved by X-ray diffraction, namely, those of NaCl and KCl.3 It had also been explored by my former mentor Monteath Robertson as a tool for determining the structure of some haem-like compounds,4 but most crystallographers at the time thought that the incorporation of even the heaviest atoms would have only a negligible impact on the X-ray diffraction of a protein crystal. Nevertheless, in 1953, Max had shown that, using heavy-metal isomorphous replacements, it was possible to determine the phases of the (h0l) reflections of his monoclinic haemoglobin crystals.5 Admittedly, these reflections were of a centric zone and therefore limited to be either 0° or 180°, but this was enough to give hope that a structure determination might be possible.

John Kendrew, a former student of Max, had initiated a study of the smaller myoglobin molecule. Myoglobin is a carrier of oxygen in muscles, much like haemoglobin is a carrier of oxygen in blood. However, myoglobin has a molecular weight of only 17 kDa and consists of a single polypeptide chain, whereas haemoglobin consists of four polypeptide chains: two α- and two β-chains each of about 17 kDa molecular weight. With his smaller protein molecule, John had made more rapid advances than Max, using the techniques Max had developed. In 1957, John had been able to obtain a 6Å resolution electron density map of sperm whale myoglobin that seemed to represent a series of bent helices,6 probably α-helices as had been proposed by Linus Pauling.7 Models of John’s myoglobin structure in a variety of different scales were everywhere in the Hut. This structure had been the big event in the year before I arrived in Cambridge and had been heralded as being imminent by Dorothy when I heard her speak in Montreal.

AUTUMN 1958

By the time I arrived in Cambridge in September 1958, Max and Ann Cullis had regained their ability to grow good crystals of monoclinic oxygenated haemoglobin and were preparing a series of heavy-atom derivatives based on the substitution of a couple of Cys amino acids with Hg derivatives. Max had determined the x and z coordinates using the (h0l) projection data he had so carefully explored over the previous six years or more. The unresolved problem was the determination of the relative y coordinates of the heavy-atom sites. This problem existed because there is no centric projection perpendicular to the unique 2-fold axis in a monoclinic space group and no defined origin as is provided by the crystallographic 2-fold axis in the (h0l) projection. John Kendrew had encountered this problem in the determination of the 6Å-resolution map of myoglobin. Perutz8 and Blow9 had made suggestions as to how this problem might be solved, but none of the proposed functions gave unambiguous solutions.

A few steps from the Hut was an old, ugly, brick building that housed the very new Mathematics Laboratory. In this building, Maurice Wilkes and his ‘boffin’ friends had put together a machine from leftover wartime radar equipment that was capable of being programmed to do all manner of fast computations. This Electronic Delay Storage Automatic Calculator (EDSAC) was arguably the first automatic stored-program electronic computer ever built. John Kendrew had used this machine to calculate the 6Å-resolution map of myoglobin. However, by the time I arrived in Cambridge, the original EDSAC had been replaced by a new, much faster, much more powerful EDSAC II computer that had just opened its doors to other selected users (meaning friends and acquaintances of college ‘dons’). EDSAC II was the brainchild of David Wheeler. It was quickly apparent to me that EDSAC II was far more sophisticated than the machine I had used in Minnesota. I read the well-written manual and started putting some of my crystallographic experience into this wonderful new machine. However, I did not have a Cambridge background and was not aware of the conventions assumed by in-group users. As a result, I was treated with some suspicion that was not dispelled until I was able to show that my programs produced correct results in an acceptably short time frame. In addition, there was a large group of more conventional crystallographers at Cambridge who distrusted the new computers, while others, like myself, advocated their use. The prevailing thought was that the human mind should be more powerful than any machine.

One of the first tasks that I set for myself was to write a Fourier synthesis program for the calculation of electron density and other crystallographic maps. I also proposed a new and simpler function to solve the ‘y-axis’ problem mentioned above. Although program testing could be performed at certain specific times during the day, large-scale computations could be performed only on allocated nights. Max and John had been given Monday nights, to be shared with the radio astronomers of Martin Ryle’s group. Thus, one Monday, maybe in November 1958, I used my new Fourier program to compute my new y-axis correlation functions. Alas, when I came to examine the results the next morning, I realised that the symmetry was wrong. Clearly, there was a mistake (maybe many) in the program.

Max and Ann were very supportive and assured me that they could wait for me to try to fix my program. Fortunately, the problem was not serious and, two weeks later, I was back on another Monday evening repeating the same calculations with a corrected program.

My delight was indeed great the next morning. The maps showed exactly what I had hoped – positive peaks for vectors between atoms in the same compound and negative peaks for vectors between atoms in different compounds10 (Figure 2.2). Furthermore, the vectors were all self-consistent. Best of all, the peaks were sky-high, leaving absolutely no doubt in differentiating between signal and noise. Now it was certain that we had the necessary heavy-atom derivatives, the data-processing procedures, and the technology to find the correct positions of the heavy atoms, all prerequisites for determining the structure of haemoglobin. Max was delighted. He brought in every person who would understand the significance of these results, starting with the local crystallographers such as Bill Cochran and then the outsiders such as Sir Lawrence Bragg, Dorothy, and Bernal (the ‘Sage’). Mostly, he would ask me for my folders containing the maps whenever he had a distinguished visitor, but it was obvious that he was happy to have had this progress. At the same time, I began to be recognised by those who had complained about my unconventional ways and techniques.

Figure 2.2. Composite view of the (FHgAc2 − FPCMB)2 correlation function between the HgAc2 and para-chloromercurybenzoate (PCMB) heavy-atom derivatives. Continuous lines are positive contours, whereas broken lines are negative contours. The Harker sections at v = 0 and v = 1/2 have positive peaks relating the symmetry-related HgAc2 sites and also the symmetry-related PCMB sites, whereas the v = 0.3 section contains negative vectors between the HgAc2 and the PCMB sites.10

WINTER AND SPRING 1959

Now we had to start thinking about phase determination given the heavy-atom sites. But we still needed to determine the parameters that defined the size of heavy-atom substitutions (occupancies and ‘temperature’ factors). For this, I wrote a least squares program based on the y-axis correlation method.

John Kendrew and Sir Lawrence Bragg had used a manual technique for determining the phases of each reflection based on David Harker’s diagram.11

Each of the about 400 reflections used in the 6Å-resolution map of myoglobin had been analysed with a ‘Harker diagram’. This was, in reality, an Argand diagram on which were drawn calculated vectors representing the structure factors of the heavy atoms given their parameters. Circles corresponding to the observed amplitudes of all the isomorphous compounds were then drawn. Where the circles intersected was the most probable phase. John and Sir Lawrence had simply guessed at what they thought was the best point of intersection of the phase circles and averaged their guesses. Now, however, we had five times as many reflections as John had had for his earlier work.

David Blow had been a graduate student of Max, although in 1958 he was in America as a postdoctoral fellow with Alex Rich, first at the National Institutes of Health and later at the Massachusetts Institute of Technology. David had spent his PhD time thinking with Francis about how to be more rigorous12 in the selection of the best phase from the Harker diagram. Dick and Bror simplified the Blow-Crick ideas to a readily programmable algorithm.13 I had helped to teach Hilary Muirhead to program EDSAC II and suggested that she should write a phasing program. I was, however, not entirely happy with Dick and Bror’s algorithm because it assumed that all the phasing errors were in the measurement of heavy-atom derivative data, with no error in unsubstituted ‘native’ data. Thus, Hilary and I somewhat modified the procedure14 – a change that most probably aided in producing the clean map that we subsequently calculated in August 1959.

Max had briefly taught me how to mount the haemoglobin crystals into glass capillaries, how to operate the Broad X-ray generators in the basement of the Austin Wing of the Cavendish Laboratory, and how to handle precession cameras with protein crystals. Among the man-made obstacles was the chief Cavendish Laboratory technician who could never understand why we needed so much film to collect our data. Persuading him to provide a new box of film was a major diplomatic feat that usually resulted in being given merely a few films at a time.

We now had all the tools and all the data I thought were necessary for producing a 5.5Å-resolution map of haemoglobin. Max and Ann had collected about five heavy-atom derivative data sets, but Max wanted to collect one more. Although, in retrospect, that can now be seen to have been an unnecessary precaution, we had no idea what we would see in the map. Thus, when we eventually saw the electron density, there was no doubt about its significance and, considering the unexpected nature of the result, it might have been met with disbelief had the map been poorer.

AUGUST 1959

Everything was ready by August and Max agreed to the calculation of the haemoglobin electron density map. Everything, that is, except for EDSAC II, which had had one of its frequent nervous breakdowns. We impatiently waited another two weeks until Max was invited to be the first user of the repaired computer. I persuaded Max to come with me to the mathematics lab that afternoon. I tried to teach him how to feed the punched paper tapes into the computer, but he was too nervous, so eventually I started the computations myself. About an hour and a half later, the map was calculated, but it still needed plotting by hand, a tedious task that would consume our computer girls for the next few days. But I did notice that there were two sections with very high density, which presumably were the haem groups in the α- and β-chains of the molecule. (The space group was C2, with molecules sitting on crystallographic 2-fold axes.) Thus, I suggested that we should explore these positions with a finer grid interval. Max agreed, and I punched a couple of quick ‘jiffy tapes’ to do this task. This time, Max was less nervous and succeeded. Unfortunately, seconds after Max had initiated the calculations, smoke started to pour out from behind the control panel, and everything closed down. Max made a comment, something like, ‘You see, I should not have operated the computer.’ EDSAC II was closed for another two weeks. Then John Kendrew, Dick Dickerson, Bror Strandberg and David Phillips (their collaborator from the Royal Institution in London) had their turn in using EDSAC II to calculate the 2Å-resolution map of myoglobin. I remember receiving a phone call late that evening from David Phillips asking me to come to the mathematics lab to sort out a problem with my Fourier program. They then took most of the night to finish their calculation.

Once the haemoglobin maps had been plotted onto a semitransparent tracing paper (Figures 2.3–2.5), Max tried to construct a cage made of Meccano parts in his office in order to display the map sections in a way that might help make the map more easily interpretable. Yet when I looked at the maps, I could see long, thin features that were surely α-helices. I really wanted to look at the maps and not wait for Max. However, maybe out of force of habit, Max started to prepare more heavy-atom derivatives in the laboratory. What happened next I kept to myself for many decades. I had been privileged to work on the haemoglobin project with Max, but it was also a project that Max had given his whole life to develop. In my enthusiasm to look at the results, I stole the final discovery from Max. When I realised what I had done, I felt terrible. I have never truly forgiven myself for my behaviour. The excitement of discovery was completely shattered for me.

Figure 2.3. The 5.5Å-resolution haemoglobin map. Section y = 1 showing long rods of density representing α-helices.

Figure 2.4. The 5.5Å-resolution haemoglobin map. Section y = 9 showing long rods of density representing α-helices.

Figure 2.5. Interpretation of the 5.5Å-resolution haemoglobin map. I used different colours to trace regions of continuous density. A change in colour represented uncertainty of the connectivity of densities between the coloured traced sections. Numbers indicate the sections on which density was found. I could recognise the two independent, fully connected chains only when this tracing had been completed. In 2004 or 2005, I gave this map, plus my model of haemoglobin, for safe keeping to the Medical Research Council Laboratory of Molecular Biology historical archives.

When Georgina Ferry was doing her research prior to writing Max’s biography, she came to interview me at Purdue. I felt it right to tell her everything I knew about Max, which would have to include these moments on the summit of Mount Discovery as a background to understanding the character of her biographical study. Some time after her visit, she sent me her presentation of those events with a request that she be allowed to use this passage in her upcoming book.15 I found her writing to be sensitive, balanced and accurate; thus, I gave her permission to use this passage in her book. For me, this was also a catharsis on a topic about which I had spent much anguish.

The helices in the maps were all connected by density permitting a trace of both the α and β polypeptide chains. I had once contemplated, long before the map was out, that there might be some similarity between myoglobin and haemoglobin because of the coincidences of molecular weights and functions. But I was quickly put down with disdain. Nevertheless, I was not overly cowed by these comments because, having traced the density chains, I immediately tried to compare the easily available three-dimensional 6Å-resolution myoglobin model with my chain tracing. The result was obvious.

I found Max in the biochemistry laboratory. Only then did I realise how much I had hurt him. With the realisation of what I had done, all desire to explore further was completely gone. Instead, Max used his outstanding experimental skills to build a model from thermally setting clay (Figure 2.6), which I would never have had the skill or imagination to do myself. This was a perfect way of representing the structure, showing not only the ‘globin fold’ but also indicating the level of error and the packing of helices. The model was beautiful! Nature was beautiful!

Figure 2.6. Max’s model of the 5.5Å-resolution map of haemoglobin.18 A thermally setting clay was molded by Max to correspond to the density on each section outlined by a chosen contour level. The different sections were then glued together. The photograph of the model is only available in black and white, but the α-chain was shown in white, the β-chain was shown in black, and the hemes were shown in red. The model referred to in the caption to Figure 2.5 was made of wood, using a thread saw to cut along the contour lines, thereby isolating the higher-density regions that were subsequently glued together. The scale was the same as what Max had used to construct his model and the same as the original maps shown in Figures 2.3–2.5.

Notwithstanding the similarity of the myoglobin structure with the α- and β-chains of haemoglobin, Max still felt it necessary to make further checks on the correctness of the structure, in particular of the site of the all-important haem groups. He noted that the Fe atoms in the centre of the haem groups should give rise to a measurable anomalous X-ray diffraction effect. Thus, given the now-established Fe atom positions and the isomorphous replacement determination of the ‘native’ phases, it would be possible to calculate the reflections that should have the largest anomalous effect and the sign of their Bijvoet difference. In this way, I determined 32 reflections that should have the largest anomalous difference. Max then asked Tony North at the Royal Institution to measure the size and sign of the Bijvoet difference for these reflections using the diffractometer that Uli Arndt and David Phillips had designed to help John Kendrew with the myoglobin data collection. Tony found that his experimental measurements agreed with my predictions for 28 of 32 listed reflections.16 This confirmation also had a further benefit, namely in determining the absolute hand of the haemoglobin map. That information could then be applied to establish the absolute hand of the 2Å-resolution myoglobin map that Kendrew’s group had just determined using the reasonable assumption that the myoglobin and haemoglobin structures have the same hand. This demonstrated that all the α-helices in the myoglobin map were right-handed. This was also a satisfactory result, as there would be fewer steric clashes of the side chains of laevorotatory amino acids with main-chain atoms in a right-handed α-helix, given the correctness of Bijvoet’s determination of the absolute hand of amino acids being laevorotatory.17

The evolutionary significance of the haemoglobin structure16,18 was not lost on anyone. Here was a verification of Darwin’s ideas at the molecular – and therefore genetic – level. Francis Crick and Jim Watson had suggested the manner in which a gene determined the amino acid sequence of a protein, although the actual genetic code was still unknown at that time. Yet it would seem to be likely that there must have been an ancestral gene that coded for a primordial globin fold from which the present-day sperm whale myoglobin and horse haemoglobin α- and β-chains had evolved by gene duplication and mutation. For me, this discovery has had more influence on my subsequent scientific direction than any other single event.

It was a fortuitous coincidence that, in the following year, the International Union of Crystallography had scheduled its triennial meeting in Cambridge. It had been three years since I had heard Dorothy talk at the previous International Union of Crystallography meeting in Montreal. What tumultuous years those turned out to be! The 1960 meeting in Cambridge was an opportunity to display our new discoveries. Max gave a great lecture in the local cinema located in the market square, the only hall big enough to house the whole meeting. (In those days, cinemas were bigger, and scientific conferences were smaller.)

AFTER AUGUST 1959

Not long after those extraordinary days in 1959, my three office-mates dispersed around the world. Dick and Larry returned to America, and Bror stayed on a few months to initiate the building of the myoglobin model in the cold basement of the Austin Wing before returning to Sweden. But the desk vacated by Larry was quickly occupied by David Blow on his return from the United States. Although he had missed two exciting years, he (as was I) was fascinated by the crystallographic implications of structure determinations that had happened in his absence. One of David’s interests as a graduate student had been the use of anomalous dispersion to aid phase determination. I had also been thinking about the use of anomalous dispersion for finding the position of anomalous scatterers.19 We found ourselves in daily stimulating discussions, producing, in the subsequent five years, a series of papers that have helped to form the foundations of modern structural biology. The topics we covered were single isomorphous replacement (SIR) and single anomalous dispersion (SAD),20 phase combination,21 and molecular replacement (MR).22–25 The genesis of these papers came largely from the haemoglobin structure determination. We asked ourselves how few derivatives would have really been necessary to solve the structure (SIR and SAD); how crystallographic structure factor calculations should be compared to multiple isomorphous replacement phasing; and whether it would have been possible to discover the relationship between the α- and β-chains of haemoglobin without actually determining the structure (MR).

David initiated a study on chymotrypsin, a study that we initially shared equally.26 Not only were we able to use all our new ideas, but the data processing also benefited from our collaboration. Whereas Dick and Bror had used a pseudo-manual/computer sorting procedure captured in a now-famous photograph (Figure 2.7), I developed a computer sorting system for the chymotrypsin studies that I later adapted and improved for use at Purdue University and in modern commercial software.27

Figure 2.7. Dick and Bror with the myoglobin sorting board. Each piece of punched tape represented a set of intensities from one line (constant h and k) of reflections on one precession photograph. The same line of reflections might have been recorded on a number of photographs, thus permitting the determination of scale factors between photographs.

Max made it clear to me that I should try to find a more permanent job somewhere else. Fortunately, there was no shortage of job offers once we had determined the structure of haemoglobin, but they were all from America. I accepted the offer from Purdue University because I felt that the head of the biology department was genuinely interested in helping me to work on the structure of proteins. My decision turned out to be right. Although for many years I missed the intense intellectual stimulus of Cambridge, challenges at Purdue allowed me to put a personal imprint on the development of my own laboratory, which might not have been possible in a more established environment. I remain enormously grateful to Max, Dorothy, David Phillips and many other English colleagues who strongly supported my work at Purdue in numerous ways, including frequent visits and nominations to prestigious awards. It is significant that 50 years after the discovery of the haemoglobin structure, we moved into the completely new Hockmeyer Hall of Structural Biology at Purdue University.

ACKNOWLEDGEMENTS

I thank Georgina Ferry for reading and correcting my original draft of this manuscript, and Sheryl Kelly for help in its preparation for publication.

1959 was both a year of joyful discovery and a year of domestic problems for me. I remain enormously grateful to Lola and Dick Dickerson for their extremely kind and generous help without which I would not have been able to do the work described in this article. Similarly, the concern of Max and Ann (Cullis) Kennedy for my welfare remains an overriding component of my memories. Although Max was not initially a fan of computerised technology, he was always willing to search for ways to satisfy my appetite for what I deemed necessary within reasonable financial limits. I also thank David Blow for the fun years we had together, as well as my two ‘computer girls’ Jill (Collard) Dawes and Angela (Campbell) Mott for their outstanding technical help and lifelong friendship. Finally and most importantly, I thank my wife Audrey for her faithful, loving support in the face of strong competition from science, my other love.

EPILOGUE

John Kendrew met J.D. Bernal when they were both in military service in south-east Asia during the Second World War. Bernal predicted to John that the great post-war challenge would be to determine the three-dimensional structure of a protein by means of X-ray crystallography. Bernal’s final advice to Kendrew was: ‘When this is over, go home to England and solve the structure of a globular protein.’ Later, after the myoglobin structure had been solved, Kendrew’s comment on this advice was: ‘And so I did.’

Perhaps neither Bernal nor John realised, even after the structure of myoglobin had been determined, that this was the very beginning of a vast scientific revolution. The structures of myoglobin and haemoglobin (Figure 2.8) were the distant rolls of thunder, unnoticed by most, of a tremendous storm that was gathering and that would completely change the biological landscape.

Figure 2.8. John and Max standing behind the forest of rods into which the first sperm whale myoglobin model was built in the basement of the Cavendish Laboratory.

After that summer of 1959, nothing much happened in structural biology for several years. The general opinion was that perhaps there would be no further protein structures any time soon. Indeed, the world had to wait until 1964 for the structure of hen egg white lysozyme, determined by David Phillips’ team at the Royal Institution in London and overseen by Sir Lawrence Bragg.28,29 Lysozyme not only had elements of β-sheets as had been predicted by Pauling,30 but it was also the first enzyme whose structure was determined. Furthermore, the structure was able to put onto a firm basis many of the previous suggestions on how an enzyme can catalyse a chemical reaction.

Over the subsequent few years, a few other enzymes, including carboxypeptidase, chymotrypsin, ribonuclease, papain, insulin, carbonic anhydrase and lactate dehydrogenase, were determined. We realised that, suddenly, there were quite a lot of data that needed to be made accessible to all. At the Cold Spring Harbor meeting in the summer of 1971 there was a small meeting that included Max Perutz, Jan Drenth, Fred Richards, Walter Hamilton and myself, which established the Protein Data Bank for the collection of all protein (and later nucleic acid) structures. The holdings of the Protein Data Bank grew only slowly at first, but the rate has increased exponentially to the point where a total of about 50,000 structures have now been deposited.* The major factors that have made this possible are the advent of synchrotron radiation for X-ray diffraction studies, freezing of crystals to greatly reduce radiation damage, and automatic computerised data processing as well as improved methods for phase determination and phase refinement – all making it possible to solve crystal structures within hours of collecting the first diffraction data.

Biological revolution is not only the speed with which structures can now be solved, but also the increasing biological significance of the structures. Structures of viruses, ribosomes (barely known to exist in 1959) and ribozymes (not dreamed of in 1959) have become available. All these varied topics have been found to have a common thread in their evolution, as it has become apparent that many biologically essential functions are based on structures that have changed little since the appearance of life on Earth. The modern crystallographer knows little about space groups or structure factors, but does know a great deal about structure and biology. A new science, called ‘bioinformatics’, has arisen – made necessary by the enormous growth of structural and gene sequence information. Indeed, structural biology has become the unifying factor of just about every aspect of biology.

In 1959, we were a small, elite group of friends who had a partial and incomplete vision of what might be the future. Today, structural biologists are on every continent in every country. The biological knowledge exhibited by the newest generation of scientists, whether physicist, chemist or biologist, is very impressive, but he or she will probably know little, if anything, about what happened in 1959.

REFERENCES

1. G. Ferry, 1998. Dorothy Hodgkin: A Life. Granta Books, London.

2. J.D. Bernal and D. Crowfoot, 1934. X-ray photographs of crystalline pepsin. Nature 133, 794–5.

3. W.L. Bragg, 1913. The structure of some crystals as indicated by their diffraction of X-rays. Proceedings of the Royal Society of London A 89, 248–277.

4. J.M. Robertson, 1935. An X-ray study of the structure of phthalocyanines: Part I. The metal-free, nickel, copper, and platinum compounds. Journal of the Chemical Society 615–21.

5. D.W. Green, M. Ingram and M.F. Perutz, 1954. The structure of haemoglobin: IV. Sign determination by the isomorphous replacement method. Proceedings of the Royal Society of London A 225, 287–307.

6. G. Bodo, H.M. Dintzis, J.C. Kendrew and H.W. Wyckoff, 1959. The crystal structure of myoglobin: V. A low-resolution three-dimensional Fourier synthesis of sperm-whale myoglobin crystals. Proceedings of the Royal Society of London A 253, 70–102.

7. L. Pauling, R.B. Corey and H.R. Branson, 1951. The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proceedings of the National Academy of Sciences of the United States of America 37, 205–11.

8. M.F. Perutz, 1956. Isomorphous replacement and phase determination in non-centrosymmetric space groups. Acta Crystallographica 9, 867–73.

9. D.M. Blow, 1958. The structure of haemoglobin: VII. Determination of phase angles in the non-centrosymmetric [100] zone. Proceedings of the Royal Society of London A 247, 302–36.

10. M.G. Rossmann, 1960. The accurate determination of the position and shape of heavy-atom replacement groups in proteins. Acta Crystallographica 13, 221–6.

11. D. Harker, 1956. The determination of the phases of the structure factors of non-centrosymmetric crystals by the method of double isomorphous replacement. Acta Crystallographica 9, 1–9.

12. D.M. Blow and F.H.C. Crick, 1959. The treatment of errors in the isomorphous replacement method. Acta Crystallographica 12, 794–802.

13. R.E. Dickerson, J.C. Kendrew and B.E. Strandberg, 1961. The crystal structure of myoglobin: phase determination to a resolution of 2 Å by the method of isomorphous replacement. Acta Crystallographica 14, 1188–95.

14. A.F. Cullis, H. Muirhead, M.F. Perutz, M.G. Rossmann and A.C.T. North, 1962. The structure of haemoglobin: VIII. A three-dimensional Fourier synthesis at 5.5 Å resolution: determination of the phase angles. Proceedings of the Royal Society of London A, 265, 15–38.

15. G. Ferry, 2007. Max Perutz and the Secret of Life. Chatto and Windus, London.

16. A.F. Cullis, H. Muirhead, M.F. Perutz, M.G. Rossmann and A.C.T North, 1962. The structure of haemoglobin: IX. A three-dimensional Fourier synthesis at 5.5 Å resolution: description of the structure. Proceedings of the Royal Society of London A 265, 161–87.

17. J.M. Bijvoet, A.F. Peerdeman and A.J. van Bommel, 1951. Determination of the absolute configuration of optically active compounds by means of X-rays. Nature 168, 271–2.

18. M.F. Perutz, M.G. Rossmann, A.F. Cullis, H. Muirhead, G. Will and A.C.T. North, 1960. Structure of haemoglobin. A three-dimensional Fourier synthesis at 5.5-Å resolution, obtained by X-ray analysis. Nature 185, 416–22.

19. M.G. Rossmann, 1961. The position of anomalous scatterers in protein crystals. Acta Crystallographica 14, 383–8.

20. D.M. Blow and M.G. Rossmann, 1961. The single isomorphous replacement technique. Acta Crystallographica 14, 1195–1202.

21. M.G. Rossmann and D.M. Blow, 1961. The refinement of structures partially determined by the isomorphous replacement method. Acta Crystallographica 14, 641–7.

22. M.G. Rossmann and D.M. Blow, 1962. The detection of sub-units within the crystallographic asymmetric unit. Acta Crystallographica 15, 24–31.

23. M.G. Rossmann and D.M. Blow, 1963. Determination of phases by the conditions of non-crystallographic symmetry. Acta Crystallographica 16, 39–45.

24. M.G. Rossmann, D.M. Blow, M.M. Harding, and E. Coller, 1964. The relative positions of independent molecules within the same asymmetric unit. Acta Crystallographica 17, 338–42.

25. E. Dodson, M.M. Harding, D.C. Hodgkin and M.G. Rossmann, 1966. The crystal structure of insulin: III. Evidence for a 2-fold axis in rhombohedral zinc insulin. Journal of Molecular Biology 16, 227–41.

26. D.M. Blow, M.G. Rossmann and B.A. Jeffery, 1964. The arrangement of α-chymotrypsin molecules in the monoclinic crystal form. Journal of Molecular Biology 8, 65–78.

27. M.G. Rossmann, A.G.W. Leslie, S.S. Abdel-Meguid and T. Tsukihara, 1979. Processing and post-refinement of oscillation camera data. Journal of Applied Crystallography 12, 570–81.

28. C.C.F. Blake, D.F. Koenig, G.A. Mair, A.C.T. North, D.C. Phillips and V.R. Sarma, 1965. Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Å resolution. Nature 206, 757–61.

29. C.C.F. Blake, R.H. Fenn, L.N. Johnson, D.F. Koenig, G.A. Mair, A.C.T North, J.W.H. Oldham, D.C. Phillips, R.J. Poljak, V.R. Sarma and C.A. Vernon, 2001. Historical perspective. How the structure of lysozyme was actually determined. In International Tables for Crystallography (M.G. Rossmann and E. Arnold, eds). F. Kluwer Academic Publishers, Dordrecht, pp. 744–72.

30. L. Pauling and R.B. Corey, 1951. Configurations of polypeptide chains with favored orientations around single bonds: two new pleated sheets. Proceedings of the National Academy of Sciences of the United States of America 37, 729–40.

* The figure of 50,000 was passed in 2008, and in 2013 stands at 90,000.

CHAPTER 3

Fifty Years After a Sabbatical at the Laboratory of Molecular Biology

Robert G. Shulman

Sterling Professor (emeritus) of Molecular Biophysics and Biochemistry, Yale University

Figure 3.1. Robert G. Shulman. Photo by Mark Bretscher.

The Laboratory of Molecular Biology has continued to have a special place in the scientific lives of those of us who came from abroad on sabbaticals. I had the pleasure of spending the 1961–2 academic year with Francis Crick and Sydney Brenner helping them, and Leslie Barnett, clean up the loose ends of their frame shift hypothesis about the DNA reading mechanism.

In the decade before coming to Cambridge I had been in the solid state physics research department of the Bell Telephone Laboratories mainly active in introducing NMR to semiconductors, superconductors and magnetic materials, but in early 1961 I stumbled onto reports that caused me to switch to the study of biological molecules. Russian scientists had reported that DNA molecules were magnetic, seemingly embodying a swarm of unpaired electron spins. I obtained some cotton-like threads of DNA from Alex Rich, a friend from post-graduate days at Caltech, and, testing them in an electron spin resonance spectrometer, found that they were indeed magnetic; the DNA sample was ferromagnetic, in that all its electrons were lined up in a magnetic field like the electrons responsible for the magnetism of iron. The hypothesis that DNA electrons were coupled thrilled me with the hope that this unexpected, and unparalleled, physical phenomenon might help explain the mysteries of biology in general and of inheritance in particular. I ran around Bell Labs telling all my friends the great news that DNA had unexpected electronic properties, but before publishing the results, it was necessary to make sure that the electrons being measured were really in the DNA molecule and not, for example, in some impurity accompanying the DNA. Soon a few elementary measurements turned up the existence of clumps of ferromagnetic impurities in the samples, little pieces of iron oxides, which further research showed were products of the DNA extraction process. This provided an explanation for the Russian publications, disproving the revolutionary hypothesis allowed by the early data. But it changed my goals; the hook had been set: I had experienced the excitement, albeit transiently, of biological research. I did some NMR studies of enhanced water relaxation when manganese bound to DNA and changed my sabbatical plans for the oncoming year at the last moment; from continuing with condensed matter physics to entering molecular biology and biophysics. The Guggenheim Foundation, of which I was a Fellow, readily accepted and even seemed to encourage this change; so I ended up going to the hut in the courtyard of the Cavendish Laboratory in Cambridge to do research for a year, on the reading of the genetic code, with Francis Crick and Sydney Brenner.

LMB AND THE GENETIC CODE

When I arrived in Cambridge with wife and child – via a physics meeting in Japan, and a ship from Yokohama to Marseilles – with a vague promise from Sydney, relayed to me by Alex Rich, that they would try to find a desk for me, I found that a desk (literally one desk) was available because, by chance, a lab technician had just been fired. At Cambridge I moved without any biochemical experience into Crick’s laboratory (which had three desks) to perform bacteriophage experiments, testing his quite abstract theory of how the genetic code was read, which had just been published as a preliminary report in Nature. In the early 1960s the very existence of a genetic code, let alone its nature, was pure conjecture. Crick’s hypothesis was that the sequence of bases in DNA was read as a triplet code where every combination of three nucleotides was the signal for a specific amino acid to be incorporated into a protein. He proposed further that the DNA code was read serially, one triplet at a time, like a magnetic tape. When an acridine molecule, acting as a mutagen, is inserted in the DNA between two adjacent base-paired nucleotides, he proposed that it is included by the reading mechanism in a triplet. In the downstream reading of the DNA each triplet, assumed to code for a specific amino acid, would be shifted by one base-pair, resulting in coding for a different amino acid.

Francis saw that in my useless, inexperienced state I should be kept far away from active research so that I could do no damage. I was quickly assigned to a region of the phage called 1589, which seemed to be the equivalent of pitching during batting practice. Although the simple experiments I did daily were at the edge of my limited understanding (‘three drops of this and 1 cc of that, then into a Petri dish followed by overnight in the oven,’ whispered Leslie as she saw me paralysed by uncertainty), the reading model being tested was a grand hypothesis based upon understanding and guesswork. After hauling plus and minus mutants around the 1589 wilderness I had a moment of great excitement when in a standing laboratory conference as to where we should go next Francis said: ‘We could always do Sydney’s experiment.’ ‘What’s that?’ I asked, and on being told that it would be to treat the mutants, called ‘barriers’ which had been putative chain terminators, with something called bU that could change one of the nucleotides coding for chain termination into a harmless nonsense mutant and thereby allow the protein chain to continue growing, I said: ‘I’ll do that.’ So I set up an agar plate, following Leslie’s directions, to test the effects of bU (which I soon learned to be bromo-uracil) and put it in the oven. The next day I found a long beard growing out of the oven; when I called Sydney over he said it was a strange bug that contaminated the plate and told me to do it over. ‘I wonder what that bug was’, I mused. To which Sydney snapped: ‘Forget it – you’re not working for a drug house.’ (And while that was good advice at the time, I’m not sure that I haven’t followed it too closely in my later career, possibly missing some serendipitous discoveries.) Done properly, the experiment supported the hypothesis that the barriers were chain terminators and the results were included six years later as a small part of a long, definitive article in the Proceedings of the Royal Society. ‘Buried there’, Francis said.1

At a time when the code was only a hypothesis, Crick’s model had proposed that a triplet code existed and furthermore proposed bacteriophage experiments to test the hypothesis about how it was read. As the results continued to support the hypothesis, Crick, more than once, walked around the lab expressing the fear that the next experiment would disprove the whole structure, so that both hypothesis and explanations would be flushed down the drain. But they survived and the frame-shift reading model remains the foundation of molecular biology.

Forty-odd years later I asked Francis why we hadn’t published these results on bU as a note in Nature or some such place. He replied, ‘We had told everyone who mattered.’

At the end of the academic year, during which time the laboratory celebrated new quarters on Hills Road with the Queen in attendance, I gave a seminar to the LMB entitled ‘The Importance of Being Manganese’, indicating my scientific intentions of returning to the start I had made in biophysical studies of paramagnetic materials by NMR. Years later, Sydney pointed out to me that in not pursuing molecular biology I was following him and Francis, both of whom moved in new directions after the frame shift mutants – he to the worm and Francis ultimately to the brain. It was around the end of that sabbatical year that I first heard Sydney saying: ‘Molecular biology is for grown-up boys.’

BELL LABS AND HAEMOGLOBIN

I returned to Bell Telephone Labs with an exciting experience of modern biology, and once there my colleagues and I were encouraged by the far-sighted management to continue biophysical research. We studied biological materials, e.g. proteins and nucleic acids, with magnetic resonance methods such as we had previously introduced into physical studies of semiconductors, superconductors and anti-ferromagnetics. Our approach was similar to that being used by the condensed matter physicists and chemists who were our colleagues. If you conduct biological research in a laboratory devoted to understanding the physics of molecules and inorganic materials, you must use the methods of physics. Furthermore, it was necessary to use the same criteria to judge the success of that effort. Bell Labs’ administration was eager to support all research efforts so long as they gave definite results. Our biophysical goals were similar; we accepted that explanations had to be based on established physical principles. The methods were traditionally empirical, but novel questions had to be formulated so as to allow physical answers in terms of energies and rates.

Soon after I returned, superconducting magnets became available for NMR and a state-of-the-art superconducting NMR spectrometer allowed me to begin the direct study of proteins and nucleic acids. My previous physical interest in paramagnetic materials (like the manganese I had considered to be important) led me directly back to research at the LMB. I started to study the paramagnetic proteins, haemoglobin and myoglobin, with Kurt Wuthrich and Seiji Ogawa and other brilliant young colleagues2 in a paper with the provocative title of ‘On the absence of “heme-heme” interactions in hemoglobin’ which disagreed with the Koshland model. Those years after my sabbatical also marked a somewhat new direction for Max Perutz who, having earlier shown different quaternary structures for both oxygenated and de-oxygenated Hb, had in 1970, after he had solved the three-dimensional structure of the two forms of Hb at atomic resolution, proposed that the structures explained the molecule’s function.3

Haemoglobin was known to perform its function of delivering oxygen to tissue by having a sigmoidal binding curve, with the binding of oxygen becoming stronger as more oxygen bound. Two theories proposed different molecular bases of this cooperative binding. One, by Dan Koshland, assumed that the binding strength increased with the number of oxygen molecules already bound, which we called heme-heme interactions. The other, by Monod, Wyman and Changeux, attributed the increase in binding strength to a switch in the quaternary structure from a T form present in the absence of oxygen, assumed to have low affinity, to a high-affinity form found when the haemoglobin was saturated with oxygen. Perutz3 accepted our results that direct steric effects were not responsible for the energy as required by Koshland’s model. He wrote of the changes of affinity shown by our NMR work: ‘Studies with mixed hybrids show no evidence that such changes occur unless there is a change in quaternary structure, which led Shulman and his colleagues to suggest that the energy of interaction arises at the boundaries between the subunits.’ He referred to references 4 and 5 (the latter of which was in press) of the present chapter. Perutz proposed3 that when oxygen bound in the deoxygenated quaternary structure it pulled the iron 0.75A down into the plane of the heme group, and that this movement was transferred to salt bridges at the interfaces between the subunits breaking them and causing the lower affinity. Since the salt bridges, across the sub-unit interfaces, only existed in the deoxygenated quaternary structure, he proposed that the unligated T quaternary structure would have low affinity in contrast to the fully ligated structure that had no salt bridges. This mechanism was his basis for linking the affinity to the quaternary structure and therefore supporting the Monod model.

In Seiji’s NMR experiments4 the number of ligands were held constant by two of the four subunits being ligated while the quaternary structure was switched by changing solution conditions. Seiji Ogawa, John Hopfield and I had expanded on the conclusion5 from Seiji’s experiments, that Hb binding was explained by the Monod model where the affinity was linked to the quaternary structure, not to the number of ligands already bound, as in the Koshland model. The ability to switch the quaternary structure was facilitated in this experiment by fixing the molecule with only two ligands bound, as it would be at an intermediate step during the binding process. The switch was identified by NMR markers of the quaternary structure, and in collaboration with Quentin Gibson we showed that the binding kinetics (and therefore the affinities) switched with the quaternary structure, while the number of ligands was held constant.6 With Seiji, John and other colleagues over the next ten years we wrote a number of papers showing that the Monod model could fit all the kinetic data published since the beginning of the 20th century. In testing Perutz’s claims about the structural proof of the Monod model my colleagues and I performed a number of experiments,7 showing that the mutant Hb Kansas, which remained in the T quaternary structure on ligation,8 showed low affinity for ligands characteristic of the T structure, even though the salt bridges did not break on ligation.8

In 1974, in the lunch line at Bell Labs, I met Peter Eisenberger and Brian Kincaid, who were excitedly discussing a new technique, extended X-ray absorption fine structure (EXAFS), that had the sensitivity to measure the bond lengths of molecules with an accuracy of a few hundredths of an angstrom. Discussion revealed that EXAFS had the sensitivity to measure Fe bond distances in proteins. We first9 studied the iron sulfur protein, rubredoxin, showing that contrary to proposals the four Fe-S were identical to within our experimental accuracy of ~0.02A. With Ogawa, Eisenberger and Brown I turned to the study of Hb10 and obtained the EXAFS spectrum showing no changes in the iron to nitrogen distances (to within 0.02A) between the low-affinity forms of deoxygenated HbA and in the high-affinity forms of deoxygenated HbKempsey (as well as no changes in the iron to ligand distances in the high and low affinity forms of fully ligated Hb). We concluded, from quantitative considerations of bond strain energy, and from the same structure around the Fe in both high and low affinity Hb, that ‘the energies responsible for the affinity differences are not located at the iron, which has been described as the absence of heme-heme interaction’.2

Improvements in EXAFS techniques and data analysis allowed us11 to determine the lengths of the iron to porphinato nitrogens Fe-Np in Hb and HbO2 as well as in the picket fence model compounds PF and PFO2, small molecule models of the heme binding site capable of binding oxygen reversibly. Triangulation of the 2.055 FeNp bond length with model compound data showed the Fe was 0.2+0.1/–0.2A out of the plane. We claimed that ‘if the iron is indeed out of the plane … it has not been forced out by the FeNp bond … nor has it been pulled out by the globin since PF and Hb are identical but probably it is moved by its uncompensated steric hindrance of the axial imidazole ligand.’

In 1987 Fermi approached me with the proposal that since high resolution X-ray data showed that the Fe-Pn distance above the plane was 0.4 A, we should write a paper together showing the reconciliation of EXAFS and X-ray crystallographic results. This I was happy to do because the converging evidence from two highly technical methods was something to be celebrated. The paper12 claimed that ‘the distance of the Fe above the plane of the porphyrin nitrogens was 0.38+/–0.04 from the crystallographic study; this value is not far from the upper limit of the distance 0.20+/–0.10/0.20A calculated from the EXAFS experiment by triangulation. These distances above the nitrogen plane are shorter than those estimated in the earliest X-ray structures.’

These issues were dormant for several years until Perutz, in collaboration with Dodson and colleagues, published ‘The stereochemical mechanisms of the cooperative effects in hemoglobin revisited’13 in which they reviewed Perutz’s 1970 stereochemical basis for the Monod model with a low-affinity tense (T) quaternary structure constrained by salt bridges and a relaxed high-affinity form without salt bridges. In summary they stated: ‘Almost every feature of this mechanism has been disputed, but the evidence that has come to light more than 25 years later now shows it to have been substantially correct.’

However, I do not believe that the data supported that conclusion. In a subsequent review14 I showed how the energetic and kinetic results from spectroscopic data (NMR and EXAFS) established the Monod model. I also summarised the crystallographic data, including haemoglobin in polyethylene glasses, in which Hb could be ligated and remain in the T quaternary structure. These results showed that the T quaternary structures had low affinity for binding, but since the salt bridges did not break upon ligation they were not the cause of low affinity.

MAGNETIC RESONANCE SPECTROSCOPY

Starting with the measured energetics of Hb and other bio-molecules my research soon moved into studying the free energies and kinetics of metabolic reactions in vivo, taking advantage of the non-invasive nature of the radio waves used in NMR experiments to follow the rates of synthesis and degradation of the small molecules, or metabolites, whose chemical reactions do the daily work of biology.

Magnetic Resonance Spectroscopy (MRS), which has been developed to a great extent in my laboratory at Bell Labs and subsequently at Yale, follows chemical reactions in the body.15 Following metabolic reactions by MRS and locating them in vivo by MRI adds new dimensions to the classical, textbook understanding of metabolism. At first the small magnet of our NMR spectrometer could only accommodate suspensions of cells in solution, primarily E. coli and yeast, but as larger superconducting magnets capable of enclosing small animals and eventually humans became available, our studies addressed intact multi-cellular organisms – eventually focusing on humans.16

The 13C nucleus is about 1 per cent abundant and it has been useful to start with 13C enriched compounds and to follow their reactions in vivo by 13C MRS. Glucose, enriched in 13C up to more than 90 per cent, can be chemically synthesised or purchased, and has been a valuable starting substrate for measuring the rates of several fundamental biochemical pathways. After developing the technique in liver, heart and skeletal muscle and showing its relevance for type 2 diabetes15 my research, with talented younger colleagues at Yale, turned to the study of energetics and neuronal activity in the rat and human brains.

BRAIN ENERGETICS AND OBSERVABLE BEHAVIOUR

When a neuron fires, doing the work of the brain, it releases a neurotransmitter which drifts across a synapse to the next neuron. The most common cortical neurotransmitter is glutamate, making up more than 90 per cent of the synapses. Early MRS studies of glucose metabolism in the brain measured the rate of 13C labelled glucose flow into the glutamate pool which, because it was coupled with intermediates of the Krebs cycle, measured the oxidative phosphorylation of glucose.16 Subsequent experiments measured the flow of labeled glutamate into glial glutamine and then back to neuronal glutamate which was the pathway of neurotransmitter flux caused by neuronal firing. Comparing the rates of glucose oxidation and of the glutamate neurotransmitter cycling showed that for every glutamate released as a neurotransmitter one glucose was fully oxidised.16,17 These quantitative results showed that in the awake, unstimulated state both rat and human brains devoted nearly all of their energy to the processes supporting glutamate neurotransmitter release and cycling. They established a reliable one-to-one relation of total brain energy consumption and the neural work done – the basic thermodynamic parameters. Since neuroimaging methods such as fMRI, positron emission tomography (PET) and 13C MRS all measure the localised brain consumption of glucose and oxygen, the one-to-one relation means they can be quantitatively analysed to determine the work of neuronal firing. This quantitative relation between neuronal firing and energy consumption raised the hopes that this physical understanding of brain activities, measured by powerful modern imaging methods, could help delineate the brain activities necessary for human behaviour.

The difference between my subsequent studies of mental processes like consciousness, memory and attention and most research on mental processes is that I haven’t tried to define these terms. The word ‘consciousness’ is an abstraction, a hypothesis, and to seek brain basis of that abstraction – like looking for the brain basis of memory, attention, free will, or love – introduces uncertainties into a physical experiment. Designing experiments to reach these goals, which are not well defined, necessarily weakens the reliability offered by the physically meaningful brain measurements. Instead my colleagues and I have defined our goals as using non-invasive NMR methods to identify brain properties that are necessary for the person to exhibit a well-defined, observable behaviour.18 To understand the diverse assumptions about mental processes that continually influence our thinking about brain functions, coming from psychology, philosophy and everyday usage, required that they be understood as manifestations of philosophical traditions.19 In an effort to clarify brain studies I have examined some factors influencing our view of the brain in a volume soon to appear entitled Brain imaging: what it can (and cannot) tell us about consciousness.20

REFERENCES

1. L. Barnett, S. Brenner, F.H.C. Crick, R.G. Shulman and R. Watts-Tobin, 1967. Phase-shift and other Mutants in the First Part of the rIIB Cistron of Bacteriophage T4. Philosophical Transactions of the Royal Society, b252:487.

2. R.G. Shulman, S. Ogawa, K. Wuthrich, T Yamane, J. Peisach and W.E. Blumberg, 1969. On the absence of ‘heme-heme’ interactions in hemoglobin. Science 165, 251–257.

3. M.F. Perutz, 1970. Stereochemistry of cooperative effect in haemoglobin. Nature 228, 726–34.

4. S. Ogawa and R.G. Shulman, 1971. Observation of allosteric transition in hemoglobin. Biochemical and Biophysical Research Communications 42, 9–15.

5. R.G. Shulman, S. Ogawa and J.J. Hopfield, 1971. An allosteric model of hemoglobin. Cold Spring Harbor Symposia on Quantitative Biology 36, 337–41.

6. R. Cassoly, Q.H. Gibson, S. Ogawa and R.G. Shulman, 1971. Effects of phosphate upon CO binding kinetics and NMR spectra of hemoglobin valency hybrids. Biochemical and Biophysical Research Communications 44, 1015–21.

7. S. Ogawa, A. Mayer and R.G. Shulman, 1972. High resolution proton magnetic resonance study of the two quaternary states in fully ligated hemoglobin Kansas. Biochemical and Biophysical Research Communications 49, 1485-91.

8. L. Anderson, 1975. Structures of deoxy and carbonmonoxy haemoglobin Kansas in the deoxy quaternary conformation. Journal of Molecular Biology 94, 33-50.

9. R.G. Shulman, P. Eisenberger, W.E. Blumberg, N.A. Stombaugh, 1975. Determination of the iron-sulfur distances in rubredoxin by x-ray absorption spectroscopy. Proceedings of the National Academy of Science of the United States of America 72, 4003–7.

10. P. Eisenberger, R.G. Shulman, G.S. Brown and S. Ogawa, 1976. Structure-function relations in hemoglobin as determined by x-ray absorption spectroscopy. Proceedings of the National Academy of Science of the United States of America 73, 491–5.

11. P. Eisenberger, R.G. Shulman, B.M. Kincaid, G.S. Brown, S. Ogawa, 1978. Extended X-ray absorption fine structure determination of iron nitrogen distances in haemoglobin. Nature 274, 30–4.

12. G. Fermi, M.F. Perutz and R.G. Shulman, 1987. Iron distances in hemoglobin: Comparison of x-ray crystallographic and extended x-r absorption fine structure studies. Proceedings of the National Academy of Science of the United States of America 84, 6167–8.

13. M.F. Perutz, A.J. Wilkinson, M. Paoli and G.G. Dodson, 1998. The stereochemical mechanism of the cooperative effects in hemoglobin. Annual Review of Biophysics and Biomolecular Structure 27, 1–24.

14. R.G. Shulman, 2001. Spectroscopic Contributions to the Understanding of Hemoglobin Function: Implications for Structural Biology. IUBMB Life 51, 351–7.

15. R.G. Shulman and D.L. Rothman, 2001. 13C NMR of intermediary metabolism: implications for systemic physiology. Annual Review of Physiology 63, 15–48.

16. N.R. Sibson, J. Shen, G.F. Mason, D.L. Rothman, K.L. Behar and R.G. Shulman, 1998. Functional energy metabolism: in vivo 13C-NMR spectroscopy evidence for coupling of cerebral glucose consumption and glutamatergic neuronal activity. Developmental Neuroscience 20 (4–5), 321–30.

17. F. Hyder, A.B. Patel, A. Gjedde, D.L. Rothman, K.L. Behar and R.G. Shulman, 2006. Neuronal-glial glucose oxidation and glutamatergic-GABAergic function. Journal of Cerebral Blood Flow and Metabolism 26, 865–77.

18. R.G. Shulman, F. Hyder, D.L. Rothman, 2009. Baseline brain energy supports the state of consciousness. Proceedings of the National Academy of Science of the United States of America 106, 11096–101.

19. R.G. Shulman, 2011). A philosophical analysis of neuroenergetics. Frontiers in Neuroenergetics 3(6), 1–7.

20. R.G. Shulman, Brain imaging: what it can (and cannot) tell us about consciousness (Oxford University Press, 2013).

CHAPTER 4

Seeing How Ligands Bind (1962–3)

Lubert Stryer

Department of Neurobiology, Fairfield Science Building, Stanford University

I first met John Kendrew in 1960, when I was completing my medical studies at Harvard. John gave an exciting and inspiring talk on his recent elucidation of the three-dimensional structure of myoglobin and what it revealed about general principles of protein structure. A Helen Hay Whitney Fellowship enabled me to spend a year with Edward Purcell in the physics department at Harvard and then come in August 1962 to the Laboratory of Molecular Biology (LMB) in Cambridge, England, to carry out crystallographic studies in John’s lab. My wife Andrea, our nine-month-old son Michael and I moved into the top floor of a pleasant house on Fendon Road, just across Hills Road from the lab. John introduced me to Herman Watson, with whom I worked closely throughout my stay at LMB. Herman and his wife, Salwa, and Ken Holmes and his wife, Mary, welcomed us to Cambridge and did much to make our stay in Cambridge rewarding and pleasurable.

In 1962, the structure of metmyoglobin (ferrimyoglobin) was known at a resolution of 2Å and all but 100 of some 1,260 non-hydrogen atoms had been located. Thus, the time was ripe for an X-ray study of the mode of attachment of ligands to its heme group. John suggested that I choose a derivative whose crystal is isomorphous with that of metmyoglobin, since this would very much simplify the elucidation of its structure. I was eager to solve the structure of oxymyoglobin, a physiologically relevant form whose crystal is indeed isomorphous with that of metmyoglobin. However, I found that crystals of oxymyoglobin autooxidised from the ferrous to the ferric state in less than an hour (fifteen years later, Simon Phillips succeeded in stabilising the oxy form of the crystal by working at low temperatures). By contrast, crystals of the azide complex of metmyoglobin proved to be stable, in addition to being isomorphous. Furthermore, this complex was of interest because the azide ion was well known to be a specific inhibitor of heme-enzymes.

My first step was to obtain an abundant supply of myoglobin for crystallisation. The freezer holding sperm whale meat, obtained years earlier off the coast of Peru, was located in the Molteno Institute on Downing Street, which was headed by David Keilin, pioneer of the study of heme-enzymes. The sperm whale meat was pitch black, a striking expression of its high content of myoglobin (~5 per cent of wet weight), all in the ferric form. I purified grams of metmyoglobin in a wet lab on the second floor, next to Allen Edmundson, who was determining its amino acid sequence. Crystallisation took place in a small room on the ground floor, near to the rotating anode X-ray generators. Michael Fuller, the laboratory steward, was most helpful in ensuring that I had the supplies and equipment needed for my research, and Herman Watson taught me how to use precession cameras.

We thought it likely that the difference Fourier method could be used to determine the mode of binding of the azide ion to metmyoglobin. For an isomorphous derivative, it should only be necessary to collect a new set of diffracted intensities; the phases of the native structure should be a sufficiently good approximation to those of the derivative. To test this idea, I measured the intensities of the h0l reflections to obtain the difference in electron density between the azide and native metmyoglobin structures as projected along the two-fold b axis. The films were scanned by Eva Rowland using a Joyce–Loebl densitometer and the intensities of the reflections were punched on Hollerith cards. About once a week, I joined other crystallographers, all laden with Hollerith boxes, in taking the train to London to use a 7090 computer at IBM’s centre near Soho to process our data. Herman usually came, as did Carl Branden, who was trying a new approach, based on treating peptide groups as rigid bodies to constrain the number of parameters, to refining the 1.4Å electron density map of metmyoglobin. We would anxiously wait near the 7090 to see whether our jobs were accepted; a single mispunch could wreck a run. Once running, we would relax and head to Soho for a leisurely lunch, before returning to IBM to pick up our boxes of cards and much paper output (if all went well), which we read on our journey back to Cambridge.

Our two-dimensional projection difference Fourier synthesis was very encouraging. It contained only two significant peaks; a positive one close to the projected sixth coordination position of the heme, the expected binding site of the azide ion, and a negative peak. We decided that it would be worthwhile to collect a complete set of three-dimensional data for the azide derivative. I did so over a period of several months, guided by Herman. John was very supportive but away from LMB much of the time. He was serving as deputy science advisor to the Ministry of Defence and also devoting much time and energy to forming what became the European Molecular Biology Organization (EMBO), a testament to his international outlook and vision. The procedures I used to collect three-dimensional data at a resolution of 2Å for the azide derivative were nearly the same as those used earlier for the native protein. Seventeen sections through the reciprocal lattice, each requiring about eighteen hours of exposure to Cu Kα X-rays from a rotating anode tube, were recorded on film using a precession camera and multiple exposures to capture the wide dynamic range. More than 7,000 reflections were densitometered. The mean absolute difference in intensity between the azide derivative and native protein was sixteen electrons, compared with a mean experimental error of about 10 electrons and a mean intensity of 200 electrons.

In late spring 1963, we calculated a three-dimensional difference map at grid points separated by about 2/3Å on an IBM 7090 at Aldermaston. We were gratified to find only two significant peaks in the three-dimensional difference Fourier synthesis. The positive peak was readily identified as the azide ion because it was located near the sixth coordination position of the heme iron atom. In metmyoglobin, the iron atom is bonded to the four pyrrole nitrogens, an imidazole nitrogen atom of the proximal histine residue (the fifth coordination position), and the oxygen atom of a water molecule at the sixth coordination position (Figure 4.1a). Our difference map showed that Nα of the linear N3-azide ion is directly bonded to the heme iron and is hydrogen bonded to the distal histidine residue. The displacement of the water O by the azide Nα is nearly isoelectronic and hence does not give rise to a detectable difference peak. The location of the positive peak corresponds to a point between the azide Nα and Nβ atoms (Figure 4.1b and 4.1c). These inferred coordinates place the bound azide nitrogen atoms 1.15Å apart, in good agreement with known inter-nitrogen distances of inorganic azides. Also, the angle of the azide group with respect to the heme matched that seen in small-molecule azide-heme derivatives.

Figure 4.1. A sketch in projection of the heme region in (a) native metmyoglobin and (c) the azide derivative. The position of the difference peaks is depicted in (b). From Stryer, Kendrew, and Watson (1964).

The negative peak in the difference map arose from the ejection of a bound sulfate ion that in metmyoglobin was hydrogen bonded to the other ring nitrogen of the distal histidine residue. In essence, the negatively charged azide ion repelled the bound sulfate and ejected it into the mother liquor. The binding of the sulfate ion in metmyoglobin is not physiologically relevant; it occurs in the crystal because of the presence of 2.8 M ammonium sulfate. However, whether sulfate is bound is a graphic indicator of the electrostatic potential surrounding the distal histidine residue. The ejection of the sulfate ion is a clear-cut example of linkage between distinct sites, in this case 9Å apart. The participation of the imidazole ring in this linkage showed the ease of conversion of its secondary and tertiary nitrogen atoms, which have very different properties that are often exploited in the catalytic mechanisms of enzymes.

Sir Lawrence Bragg visited LMB during the summer of 1963. Herman and I had the privilege of showing him our difference Fourier map and discussing our interpretation of it. With a twinkle in his eye and a chuckle he said, ‘Ah, very good, you’re returning to seeing just a few atoms.’ I will always treasure meeting Sir Lawrence and seeing his delight in observing the flourishing of a field he founded and nurtured for more than half a century.

Our study of azide metmyoglobin demonstrated that interactions of proteins with small molecules can readily be detected by the difference Fourier method using intensities from the derivative and phases for the native protein.1 The difference peak heights are approximately half of what would be obtained using the precisely correct phases of the derivative instead of approximately correct phases of the native protein, clearly sufficient to readily detect the insertion of two nitrogen atoms. We concluded that the mode of binding of coenzymes, substrates and inhibitors to enzymes should be amenable to study by the difference Fourier method, provided that the derivatives are isomorphous. This promise was soon realised, as exemplified by crystallographic studies of chymotrypsin2 and lysozyme3 containing bound inhibitors. It was also gratifying to see the difference Fourier method used to solve the structure of deoxymyoglobin4 and azide methaemoglobin.5 Indeed, a difference Fourier map can detect much smaller features of electron density than are evident on a normal Fourier map calculated with the same phases.6

My stay at LMB was greatly enriched by having tea or coffee in the canteen twice a day. The spontaneous scientific interchanges there were vibrant and highly informative. I have vivid memories of learning about the colinearity of gene and protein from Tony Stretton, the nature of the genetic code from Francis Crick, amber nonsense mutations from Sydney Brenner, and the molecular architecture of muscle filaments from Hugh Huxley. The hall and offices of the Structural Studies Division were another lively venue. I learned much about virus structure and assembly from Aaron Klug and caught frequent glimpses of intense discussions between Michael Rossmann and David Blow that led to their ‘rocket’ for phase determination. It was a time of great ferment.

Andrea and I enjoyed our social interactions with the LMB community. The MolBiol Players (the name we gave ourselves to qualify for the borrowing of multiple copies of plays from the city library) had fun reading plays by Shaw, Ibsen and Anouilh. We also happily recall a wine tasting at Peterhouse, which began with a mediocre ‘workers’ wine’ and progressed in six or so stages to a premier cru from Bordeaux. Andrea and I learned quickly but, alas, have since then savoured few wines of the quality of Peterhouse’s top selections. Andrea and I also remember an evening in London, where we saw Paul Scofield in a remarkable performance of King Lear, the most riveting we have ever seen. Ken and Mary Holmes enabled us to partake in the May Ball at St John’s, where we were also joined by Reuben and Pat Leberman, and by Sam and Marilyn Krimm, in a magical evening and night that ended with the soft light of early dawn.

In the spring of 1963, John told me that he had received a letter from Arthur Kornberg at Stanford saying that they had an opening for an assistant professor and inviting him to suggest candidates. John asked me whether I would like to be considered for a position there. After a few days thought I said yes, and several weeks later received an invitation from Arthur to visit the Department of Biochemistry. At the end of my visit to Stanford, Arthur offered me a position in the department. Though I had planned on staying at LMB for two years, I eagerly accepted Arthur’s very attractive offer. After submitting the azide metmyoglobin manuscript to the Journal of Molecular Biology, I went with Andrea and Michael to Italy for sightseeing and the International Congress of Crystallography, which was held in Rome. The protein crystallography session there was exciting, full of promise. We bade farewell to Herman, Carl, our other friends from LMB, and several other protein crystallographers at a warm parting dinner at a trattoria in the Piazza Navona. This celebration marked the end of an era in which protein crystallographers knew each other and essentially everything that was happening in the field, and the beginning of a new one in which the full power of structural biology came into its own to dramatically transform the life sciences.

REFERENCES

1. L. Stryer, J.C. Kendrew and H.C. Watson, 1964. The mode of attachment of the azide ion to sperm whale metmyoglobin. Journal of Molecular Biology 8, 96–104.

2. P.B. Sigler, B.A. Jeffery, B.W. Matthews and D.M. Blow, 1965. An X-ray diffraction study of inhibited derivatives of alpha-chymotrypsin. Journal of Molecular Biology 15, 175–92.

3. L.N. Johnson and D.C. Phillips, 1965. Structure of some crystalline lysozyme-inhibitor complexes determined by X-ray analysis at 6 angstrom resolution. Nature 206, 761–3.

4. C.L. Nobbs, H.C. Watson and J.C. Kendrew, 1966. Structure of deoxymyoglobin: a crystallographic study. Nature 209, 339–41.

5. M.F. Perutz and F.S. Mathews, 1966. An X-ray study of azide methaemoglobin. Journal of Molecular Biology 21, 199–202.

6. R. Henderson and J.K. Moffat, 1971. The difference Fourier technique in protein crystallography: errors and their treatment. Acta Crystallographica B 27, 1414–20.

CHAPTER 5

From MRC-Cambridge to Madison, Wisconsin

William Dove and Alexandra Shedlovsky

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI

Figure 5.1. Bill Dove.

Figure 5.2. Alexandra Shedlovsky.

PROLOGUE – A STYLE OF DOING SCIENCE

Our experience in the early 1960s as postdoctoral fellows in the MRC Unit for Molecular Biology led to our understanding the following principle: science is effectively driven by an idea when the seed is nourished by dialogue between contrasting outlooks. We shall share our experiences that illustrate this ‘core principle’ by which the MRC unit thrived in that era. Since science does not function in a vacuum we include our perceptions of the cultural context that stimulated the growth of ideas through dialogue. This dialogue led to the remarkable edifice of molecular biology that emerged from the unit and its progeny around the world. Our comments are strictly matters of personal perception; however, Horace Judson’s Eighth Day of Creation gives a more global view of this and surrounding eras,1 while Sidney Altman wrote from a personal perspective of his experiences in the unit a decade later [‘RNA processing: a postdoc in a great laboratory’2]. Finally, the core principle is revealed in the essay written by the physical chemist John Platt [‘Strong Inference: Certain systematic methods of scientific thinking may produce much more rapid progress than others’3].

The messenger RNA experiments, the demonstration of the ‘triplet code’, and Marshall Nirenberg’s polyU encoding polyPhe experiment were all published by the middle of 1961. Alexandra arrived at the MRC Unit while it was housed in the Cavendish Laboratory of Physics and Bill came in the autumn of 1962 after the move to Hills Road. A ‘Crick Week’ was established to present all of the emerging information and stimulate debate on the current status of the coding problem. Crick focused on the steps needed to establish the genetic code, while Brenner believed that the important issues involved regulation of genetic information, including the host-induced modification of genomes.

Bill’s Caltech experience with Linus Pauling (on clathrates formed by general anaesthetics) fed into his frequent discussions in the canteen with the crystallographers. At the time, Bill sensed that any connection with Linus Pauling was cause for tension. Jim Watson’s mea culpa [‘The Double Helix’4] explains this fear of competition from Caltech’s eminent structural chemist, a founder of ‘molecular biology’. This nascent discipline, consolidated by the newly established Journal of Molecular Biology with John Kendrew as editor, radiated throughout academia. Departments of ‘Molecular Biology’ were set up across the United States, sometimes over the objections of the pre-existing biological departments. Francis Crick once quipped that soon there would be ‘departments of molecular theology’.

In the early 1960s scientific discourse in the MRC unit involved daily debate, both between the structural and the functional analysis of biology and between the genetic code and the regulation of its usage. Ideas were tested in this crucible, but put into action in the hands of the group leaders and their members who carried out experiments. Tony Stretton (on the staff) and Anand Sarabhai (a graduate student) provided the hard work needed to establish the colinearity of the major coat protein of phage T4 and its genetic map. Roger Freedman, a doctoral student with Brenner, illustrated the synergy between creative thinking and active experimentation. At a conference that he attended with Bill, Roger recognised a way to test the Campbell model for insertion of a prophage into the bacterial chromosome. Roger’s idea was proven at Stanford some years later [‘The linear insertion of a prophage into the chromosome of E. coli shown by deletion mapping’5] . Similarly, it was Roger, with Simon Pickvance, who recognised that the somatic division lineages of the nematode C. elegans could be followed in vivo by Nomarski interference optics; in this case the hard work was done by John Sulston, Bob Horvitz and others [‘The Joy of the Worm’6]. Overall, in the MRC Unit for Molecular Biology, science was driven by ideas that were nourished or rejected by discussion and brought to fruition by rigorous experiments.

ECHOES ALONG THE CORRIDOR OF RESEARCH

Bill’s secondary school, Phillips Andover, has the motto Finis origine pendet (‘The end depends on the beginning’). As we reflect on the path of research that we have followed over the past decades, we can discern echoes from the MRC unit of 1962–3. A major theme is that of mutagenesis. With his doctorate in chemistry, Bill was initially attracted to the MRC by the frameshift experiment that established the triplet code [‘General nature of the genetic code for proteins’7]. This experiment rested on the chemical evidence from Leonard Lerman that the mutagen proflavine differed in action from alkylating mutagens by intercalating into the double helix. The research in mouse and rat genetics and cancer biology that we have developed over time has rested on an understanding of the mutagenic mechanism of the directly acting alkylating agent ethylnitrosourea (ENU) [‘Induction of recessive lethal mutations in the T/t-H-2 region of the mouse genome by a point mutagen’8]. This echo currently reverberates in interesting ways with sources from the ’60s. We collaborate with investigators at the Sanger Institute to establish ENU-induced derivatives of a standard inbred mouse strain to permit genetic mapping under isogenic conditions. Curiously, this idea reflects the isomorphous replacement strategy of Max Perutz. And when Bill talks at the Fred Sanger Institute, it is in the Francis Crick Lecture Theatre!

Brenner’s choice to concentrate on issues in genetic regulation at the time that the genetic code was being finalised led to his formulation with Francois Jacob of the Replicon Model [‘On the regulation of DNA replication in bacteria’9]. Bill’s research on replication control in phage lambda [‘Positive and negative control of bacteriophage lambda DNA replication’10], the syncytial eukaryote Physarum polycephalum [‘Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum’11], and in intestinal tumours of the mouse [‘A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse’12] and rat [‘A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer’13] has used mutagenesis to dissect replication control systems of increasing complexity.

Finally, the most distant source from MRC 1962 is echoed in a most recent investigation from our laboratory: the epigenetic modification of the genome. Alexandra worked with Sydney to demonstrate that the DNA of phage T4 is subject to a form of host-induced modification – the glycosylation of hydroxymethyl cytosine residues [‘A chemical basis for the host-induced modification of T-even bacteriophages’14]. Today we study the pathways by which the ‘gatekeeper gene’ (Apc) loses function in colonic neoplasms (‘The many ways to open the gate to colon cancer’15]. We have found that one pathway involves epigenetic silencing [‘Monoallelic silencing and haploinsufficiency in early murine intestinal neoplasms’16].

EPILOGUE – LEAVING TIME FOR TEA

It is striking that in the 1960s scientific progress at the MRC unit was fostered by a tradition unique to English life. The geneticist Frank Stahl commented that J.B.S. Haldane’s method to find a mathematical function to generate a genetic map ‘left time for tea’. So it was that the canteen provided a platform for discussion. Such opportunities for planned or spontaneous conversation come up in English life five times each weekday – breakfast, morning coffee, lunch, afternoon tea and supper. Even on Saturday mornings, lab denizens would congregate in the kitchen over coffee to hear Sydney Brenner free-associate about life and experiments. Americans who recognised the power of this cultural pattern tried more than once to transplant it across the Atlantic. But, to the best of our knowledge, it never took root overseas.

Scientists everywhere nourish their creative lives by fostering a passion outside the laboratory. The Cambridge scene gave each of us such an opportunity in the realm of music. Bill joined the Cambridge Morris Dancers, exploring on weekends the countryside of Cambridgeshire and beyond. Alexandra, while at the Cavendish, would plan her experiments to permit going to Evensong at the nearby King’s College Chapel. The rich musical life in Cambridge continues and our visits over the years always include concerts – sometimes with local composers in the audience.

The parallel life outside the laboratory, dubbed ‘episcience’, involved more than these cultural aspects of Cambridge life to which we became attached. The scene included a rich array of parties. Bill’s stay at 8 Scroope Terrace, in a house occupied by students from the continent and presided over by ‘Pop Prior’, featured many such gatherings. Famously, the Cricks hosted many a ‘wild’ party. After his Nobel Prize award, police were called to reduce the noise and Francis was required to explain the cause of the celebration. Hildegard Lamfrom (now remembered by a named professorship at Oregon State University) looked for any excuse to host a party: a visiting speaker, an exciting result, or … just for fun. The MRC unit of the early ’60s showed the power of camaraderie.

Our own personal and professional trajectories were seeded within this dynamic scientific and cultural environment. They became entwined when we married in 1964 and soon after moved to the University of Wisconsin in Madison. Some aspects of the scientific life of the MRC unit have resisted transplantation. What persists for us is the excitement of joining together molecular and biological ideas, and the pleasure of living in a small but culturally rich city.

REFERENCES

1. H.F. Judson, 1980. The Eighth Day of Creation: Makers of the Revolution in Biology. Simon & Schuster: 1st Touchstone edition.

2. S. Altman, 2003. Genetics 165, 1633–9.

3. J.R. Platt, 1964. Science 146, 347–53.

4. J.D. Watson, 2012. The Annotated and Illustrated Double Helix, Simon & Schuster.

5. N.C. Franklin, W.F. Dove and C. Yanofsky, 1965. Biochemical and Biophysical Research Communications 18, 910–23.

6. H.R. Horvitz and J.E. Sulston, 1990. Genetics 126, 287–92.

7. F.H. Crick, L. Barnett, S. Brenner and R.J. Watts-Tobin, 1961. Nature 192, 1227–32.

8. A. Shedlovsky, J.L. Guenet, L.L. Johnson and W.F. Dove, 1986. Genetics Research 47, 135–42.

9. F. Jacob, S. Brenner and F. Cuzin, 1963. Cold Spring Harbor Symposia on Quantitative Biology 28, 329–48.

10. M.E. Furth, J.L. Yates and W.F. Dove, 1979. Cold Spring Harbor Symposia on Quantitative Biology 43, 147–53.

11. T. Schedl, T.G. Burland, K. Gull and W.F. Dove, 1984. Journal of Cell Biology 99, 155–65.

12. A.R. Moser, H.C. Pitot and W.F. Dove, 1990. Science 247, 322–4.

13. J.M. Amos-Landgraf, L.N. Kwong, C.M. Kendziorski, M. Reichelderfer, J. Torrealba, J. Weichert, J.D. Haag, K.S. Chen, J.L. Waller, M.N. Gould and W.F. Dove, 2007. Proceedings of the National Academy of Sciences of the United States of America 104, 4036–41.

14. A. Shedlovsky and S. Brenner, 1963. Proceedings of the National Academy of Sciences of the United States of America 50, 300–305.

15. J.M. Amos-Landgraf, L. Clipson, M.A. Newton and W.F. Dove, 2012. Cell Cycle 11, 1261–2.

16. J.M. Amos-Landgraf, A.A. Irving, C. Hartman, A. Hunter, B. Laube, X. Chen, L. Clipson, M.A. Newton and W.F. Dove, 2012. Proceedings of the National Academy of Sciences of the United States of America 109, 2060–65.

CHAPTER 6

Structural Studies of α-Chymotrypsin

Brian W. Matthews

Institute of Molecular Biology, 1229 University of Oregon, Eugene, OR

BACKGROUND

I took my PhD during 1961–63 in the Physics Department at the University of Adelaide – the same department of which W.H. Bragg had been head and his son, W.L. Bragg, a student. My initial thesis project, under the joint direction of Harry Medlin and Stan Tomlin, was to determine the crystal structure of a small molecule of fourteen atoms. At first all calculations had to be done by hand, but the university acquired a small IBM computer which I learned to program and could use to determine the structure of a more complicated complex as part of my thesis. Wanting to tackle more challenging problems, in early 1963 I wrote to Max Perutz and was offered a staff position in the MRC Laboratory of Molecular Biology in Cambridge. At that time only a single high-resolution structure of any protein was known, namely that of myoglobin determined in 1960 by Kendrew and his group. Max Perutz had spent his scientific career showing how it would be possible to determine the structures of proteins and at that point had a relatively low-resolution structure of horse haemoglobin.

On arriving in Cambridge in November 1963, and with some trepidation, I introduced myself to Dr Perutz. He explained that in the interim since he had written to offer me the position, two other postdocs had joined his group. If I still wanted to work with him I could do so, but he strongly encouraged me to spend some time reading and meeting other group leaders with the possibility of joining another group. It might be noted in passing that it is indicative of Max’s attitude toward research that he had no desire to create a large research team. Rather he preferred to supervise a small group and to have time to continue his personal ‘hands-on’ research activities.

The group I joined was that of David Blow. David’s primary research interest was the crystallographic analysis of α-chymotrypsin, using a crystal form first obtained by Bernal, Fankuchen and Perutz. David had also collaborated closely with Michael Rossmann in the development of ‘molecular replacement’ techniques. Using this approach they had obtained evidence suggesting that the asymmetric unit of the α-chymotrypsin crystals contained two α-chymotrypsin molecules related to each other by a non-crystallographic twofold axis of symmetry (Blow et al, 1964). Furthermore, they had also determined the apparent position of this twofold symmetry axis in the crystals. At the time that I arrived in Cambridge, Michael Rossmann was about to leave to start his own group at Purdue University.

THE TWINNING PROBLEM

Crystals of α-chymotrypsin were relatively easy to grow, but had a major problem; namely, they were often twinned (i.e. groups of molecules could have two alternative alignments within the same crystal – see Plate I of Blow et al, 1964). The presence of twinning could not be ascertained by inspection of the crystals under a microscope. Therefore, one had to mount each crystal and take preliminary X-ray data to determine the presence and degree of twinning. The larger the crystal, the more likely that it would be twinned. This made the collection of high-resolution data, which required larger crystals, almost impossible.

Barbara Jeffery, a technician working under David’s supervision, had been trying to obtain heavy-atom derivatives by binding different active site-directed ligands. At that time, the standard protocol was to mix the ligand with the protein in solution and then grow crystals of the putative complex. In order to increase the solubility of the ligand, David suggested that 2 per cent dioxan should be added to the crystallization cocktail. Unexpectedly, this completely eliminated twinning. It became possible to routinely grow high-quality crystals of the protein 0.4 mm or so in each direction, which would be very suitable for high-resolution data collection. (Much later the dioxan was shown to bind in the active site cleft, and was also close to the non-crystallographic twofold axis. This led to a type of statistical averaging which eliminated twinning.)

This serendipitous finding effectively transformed the project. It was now possible to contemplate collecting 2Å-resolution data for α-chymotrypsin, as had been used for myoglobin, the only known protein structure which could be used as an example. One of my first major tasks in the group was to collect the data for the native protein. More or less by default it was decided to collect the data photographically. A suitable diffractometer was not available, whereas the lab had multiple rotating anode generators which were maintained by superb technicians. Photographic data collection had been successful for myoglobin – although the task would be three times as great for the larger α-chymotrypsin molecule with two copies in the asymmetric unit. I settled into a routine in which a new crystal would be mounted each morning and carefully aligned with preliminary X-ray photographs. The data-collection precession photograph would then be exposed for a little less than 24 hours so that it could be developed the following morning. The crystal was then discarded and a new one mounted to be used for the next photograph. As noted in David Blow’s CIBA Medal Lecture (Blow, 1969), a 2Å-resolution data set required the equivalent of a year’s effort from a highly dedicated assistant to scan the films and enter the data onto IBM punched cards (one card per reflection).

Photographic data collection did have its advantages. In the first place each precession photograph could be visually checked as it was taken, and rejected if of insufficient quality. The centrosymmetric (h0l) zone was especially useful to check potential heavy atoms and to evaluate ligand binding (see below). Likewise the non-centrosymmetric (0kl) zone could be used to evaluate information from anomalous scattering. A method of ‘local scaling’ was developed to try to reduce systematic errors which can occur with photographic intensity measurement (Matthews and Czerwinski, 1975).

HEAVY-ATOM DERIVATIVES

As part of their prior low-resolution analysis of the α-chymotrypsin crystals, David, together with Michael Rossmann and Barbara Jeffery, had identified (PtCl4)2– as a possible heavy-atom derivative (Blow et al, 1964). To some degree this and other possible heavy-atom derivatives could be analysed in the centrosymmetric (h0l) projection. By using higher-resolution, projection data, and adapting ‘difference’ methods from small molecule crystallography (cf. Blow and Matthews, 1973), it was possible to better resolve the individual platinum sites which in turn led to some improvement in the phasing statistics.

At around the same time, David also suggested that I should try to react diisopropylfluorophosphate (DFP) with the protein. Using the h0l phase angles determined from the (PtCl4)2– derivative, I calculated the map showing in projection the difference between the DFP-reacted crystals and the native protein. The result, shown in Figure 6.1, was dramatic. There were two peaks, well above background, which presumably corresponded to the electron-dense phosphorus atoms bound in the active sites of two chymotrypsin molecules. Furthermore, as David immediately recognised, the two sites were equidistant from the non-crystallographic local twofold axis which he and Michael Rossmann had inferred from their molecular replacement analysis. This result was highly encouraging on multiple fronts. First, the clarity of the difference electron density map indicated that the phase angles determined from the (PtCl4)2– derivative were of high quality. Second, the result confirmed David and Michael’s predictions from their molecular replacement analysis and helped validate this approach. Third, the result showed the locations of the respective α-chymotrypsin active sites in the crystal.

Figure 6.1. Electron density map showing, in projection, the difference between DFP-inhibited and native α-chymotrypsin (see text for details). The two prominent peaks marked ‘x’ show the positions of the bound ligand; the straight dashed line indicates the axis of twofold symmetry which relates the two α-chymotrypsin molecules in the asymmetric unit. The map was contoured and the figure prepared by Jill Dawes (née Collard), a superb technical assistant (then referred to as a ‘computer girl’) who bore the brunt of the labour associated with photographic data collection (from Sigler et al, 1966).

PAUL SIGLER

In mid-1964, Paul Sigler joined the group. One could not wish for a more enthusiastic, energetic colleague. Paul was already a fully-qualified MD, but as a Helen Hay Whitney Fellow joined David’s group as a PhD student. Working with David Davies at the National Institutes of Health, he had shown that benzene sulfonyl fluorides could be used not only to label the active site of γ-chymotrypsin but also to generate useful heavy-atom derivatives. His initial focus was to apply the same approach to crystals of α-chymotrypsin (Sigler et al, 1966). This led to the use of p-iodobenzenesulfonyl fluoride and toluene-p-sulfonyl fluoride as a ‘matched pair’ of heavy-atom reagents. Although they are differentiated by an iodine atom, which is not especially electron dense, this is offset by their close isomorphism.

PHASE DETERMINATION

Anticipating the future calculation of a three-dimensional electron density map, David asked me to write a general purpose phase determination program. Because David, together with Francis Crick, had written the seminal paper on the subject (Blow and Crick, 1959), it would have been straightforward for him to write the necessary software. Presumably he felt it would be useful for me to become familiar with the approach.

Shortly thereafter, David gave me a copy of a preprint of an article from Tony North dealing with the phase determination from both isomorphous replacement and anomalous scattering information (North, 1965). In reading this report, it seemed that the problem could be formulated in a somewhat different way (Matthews, 1966a). Furthermore, I also began to appreciate that there were many ways in which the information from anomalous scattering measurements was complementary to that obtained from isomorphous replacement. Whenever isomorphous replacement gave marginal or weak information, anomalous scattering gave strong, definitive information, and vice versa. By systematically combining the isomorphous replacement and anomalous scattering information, one could improve techniques for both locating heavy-atom derivatives and also using these in phase determination (Matthews, 1966b). When the above work was written up, it would have been entirely appropriate for David to be a coauthor but he firmly declined. In all things he was extraordinarily generous.

DAVID PHILLIPS AND HEN EGG-WHITE LYSOZYME

Early in 1965, David Phillips came to the MRC to give a report on his group’s structural studies of hen egg-white lysozyme, which had been under way for some time. As was soon apparent, the structure had been solved! The second high-resolution structure of any protein and the first structure of an enzyme. It was a dramatic moment and tremendously encouraging for those of us working on other proteins.

Shortly thereafter, David gave a formal ‘Friday Evening Discourse’ at the Royal Institution in London. Models of the lysozyme structure were shown both in the folded form and as an extended chain reaching to the ceiling. Coloured lights had been added to highlight the locations of different types of residues. Following the lecture, we clustered around representative sheets of the electron density map mounted on light boxes. Herman Watson, who had extensive experience building the myoglobin model, immediately recognised α-helices, disulphide bridges, and larger side-chains. David Blow, Paul Sigler and I, who were standing nearby, were completely clueless. There was nothing that we immediately recognised. It was obvious that we needed to gain experience in electron density map interpretation if we were to solve the α-chymotrypsin structure. David Phillips kindly invited the three of us to spend a couple of days at the Royal Institution analysing a set of hen egg-white lysozyme maps which had been calculated at representative resolutions. This training turned out to be indispensable.

THE HIGH-RESOLUTION ELECTRON DENSITY MAP

During the latter part of 1966, we calculated the first 2Å-resolution electron density map for α-chymotrypsin. Although one could follow limited sections of the apparent polypeptide chain, it was impossible to interpret the overall structure. Richard Henderson joined the group as a new PhD student. In the meantime, my wife and I, together with our young daughter, had made plans to sail to Australia at the end of the year, spend Christmas with our relatives, and then continue by ship to the States to join David Davies’ group at the NIH.

Following our departure, Paul and Richard collected data for a further heavy-atom derivative and this made the difference. In a single evening David was able to follow the overall course of the polypeptide chain. In writing up the work, and with characteristic generosity, he designated me as first author (Matthews et al, 1967).

Even though the structure had been published, David was still concerned that there might be errors in some aspects of the structure. Therefore, he proposed that the electron density should be replotted parallel to a different cell face and that I should, as far as possible, reinterpret the map and build a model in David Davies’ lab at the NIH. David Davies very kindly agreed to this arrangement. Having the opportunity to independently interpret the electron density map was, among other aspects, a truly invaluable training experience. Also building the model did lead to some new insights. In particular, by mapping the backbone-backbone hydrogen bonding interactions, it became apparent that the overall α-chymotrypsin structure is comprised of two similar β-barrels (Blow, 1969).

SOLVENT CONTENT OF PROTEIN CRYSTALS

After I had been working with David Blow for some time, he announced that ‘if I was to be a protein crystallographer I needed to learn how to grow crystals’. Without further ado, he introduced me to Larry Smillie from the University of Alberta, who was spending a sabbatical leave at the MRC with Brian Hartley. Brian had determined the amino acid sequence of α-chymotrypsin and Larry was working on the related sequence of chymotrypsinogen B. Larry had a large container of lyophilised protein, ladled me out a few spoonfuls, and wished me well. Subsequently I was able to obtain very satisfactory crystals in two different forms. As I was about to leave his group, David Blow suggested that I should at least write a short report summarising the crystals which I had obtained.

The note was written after I had left the MRC and was submitted to Acta Crystallographica. Because I no longer had access to the crystals, I could not make crystal density measurements as was often done at the time. Instead, I checked the literature for related crystals and on this basis stated the likely number of molecules in the unit cells of the two different crystals that I had obtained. The referee, however, was highly critical of this ‘comparison’ procedure. This prompted me to make a complete survey of the solvent content of all of the protein crystals that were available at that time. This survey was then written up independently and submitted to the Journal of Molecular Biology as the ‘VM’ paper (Matthews, 1968a; see also Matthews, 1968b). As I think most readers will know, this is far and away the most heavily cited paper I have written. Sometimes critical referees can have valuable comments!

EPILOGUE

I was enormously fortunate to have the opportunity to become a member of the MRC lab, not only because of the training that I received, but also to make lifetime friendships and to see extraordinary individuals making extraordinary accomplishments. At the MRC everyone was treated equally. In the cafeteria, if a seat was free next to Francis Crick or Sydney Brenner or Fred Sanger or anyone else, you were welcome to sit and join the conversation.

Max Perutz invited me to the lab but then gave me complete freedom to join any group of my choosing. I worked with David Blow as a postdoc, but on multiple occasions David went out of his way to give me opportunities to learn different aspects of structural biology which would be of value years after I had left his group. On one occasion he had been invited to a three-week training course on scenic Lake Como. The course was intended to introduce the elements of biochemistry, genetics and molecular biology to individuals whose background was in physics or mathematics. David felt that I would benefit more from the course than he, and made it possible for me to attend. It was my first opportunity to meet the likes of Cyrus Levinthal, Wally Gilbert, Salvador Luria and Sid Bernhard. David was also extraordinarily generous in having me publish results under my own name whereas in every respect he could have been a coauthor.

Finally, I could not have been more fortunate in joining the MRC when I did. Prior to my arrival, myoglobin was the only high-resolution protein structure known. While at the MRC I heard David Phillips announce the determination of the second, hen egg-white lysozyme. Then, in 1967, we were able to report the structure of α-chymotrypsin which, together with ribonuclease A and carboxypeptidase A, became the third, fourth and fifth structures. Protein crystallography had arrived!

REFERENCES

Blow, D.M. 1969. The study of α-chymotrypsin by X-ray diffraction. Biochemical Journal 112, 261–8.

Blow, D.M. and F.H.C. Crick, 1959. The treatment of errors in the isomorphous replacement method. Acta Crystallographica 12, 794–802.

Blow, D.M. and B.W. Matthews, 1973. Parameter refinement in the multiple isomorphous-replacement method. Acta Crystallographica A29, 56–62.

Blow, D.M., M.G. Rossmann and B.A. Jeffery, 1964. The arrangement of α-chymotrypsin molecules in the monoclinic crystal form. Journal of Molecular Biology 8, 65–78.

Matthews, B.W. 1966a. The extension of the isomorphous replacement method to include anomalous scattering measurements. Acta Crystallographica 20, 82–6.

Matthews, B.W. 1966b. The determination of the position of anomalously scattering heavy atom groups in protein crystals. Acta Crystallographica 20, 230–39.

Matthews, B.W. 1968a. Solvent content of protein crystals. Journal of Molecular Biology 33, 491–7.

Matthews, B.W. 1968b. Some crystal forms of bovine chymotrypsinogen B and chymotrypsinogen A. Journal of Molecular Biology 33, 499–501.

Matthews, B.W. and E.W. Czerwinski, 1975. Local scaling: A method to reduce systematic errors in isomorphous replacement and anomalous scattering measurements. Acta Crystallographica A31, 480–87.

Matthews, B.W., P.B. Sigler, R. Henderson and D.M. Blow, 1967. Three-dimensional structure of tosyl-α-chymotrypsin. Nature 214, 652–6.

North, A.C.T. 1965. The combination of isomorphous replacement and anomalous scattering data in phase determination of non-centrosymmetric reflexions. Acta Crystallographica 18, 212–16.

Sigler, P.B., B.A. Jeffery, B.W. Matthews and D.M. Blow, 1966. An X-ray diffraction study of inhibited derivatives of α-chymotrypsin. Journal of Molecular Biology 15, 175–92.

CHAPTER 7

Z-bands, X-rays and the Flight Muscle of Giant Waterbugs (1963–6)

Michael Reedy

Department of Cell Biology, Duke University Medical Center, Durham, NC

Figure 7.1. Mike Reedy, 1965.

Seattle, 1957, my first term in medical school, a second-year housemate flagged me down: ‘Hey Reedy, remember you told me Prof. H. Stanley Bennett would let you try electron microscopy on skeletal muscle to evaluate his idea that insulin might affect sugar uptake by stimulating micropinocytosis? You should go to the 4 o’clock seminar today. English visitor from London, Hugh Huxley, had great EM pictures of muscle in his new paper last month.’ I went, and came away very excited, both by Huxley’s EMs that I found totally and directly convincing as arguments for the ‘sliding filament’ model of sarcomere length changes, and by the lively implications the images suggested about the structure and actions of the two kinds of myofilaments and the cross-bridges connecting between them. Five years later, now an MD, still at the University of Washington in Seattle, just about a month before the Cuban-Soviet missile crisis, I put aside autopsy pathology for an hour to hear an exciting update seminar by Huxley about isolated single molecules and filaments of actin and myosin. We met the next day, the meeting arranged by Dr John Luft, the inventor of Epon embedding mixtures and mentor of my student research, and I got to tell Hugh my discoveries about the Z-band in rat diaphragm muscle.

My research had switched to myofibril structure when the EM showed no visible membrane response to insulin. To my hair-raising delight, I’d then discovered, all in one night, that thin filaments, having emerged from a hexagonal A-band lattice where they overlapped with thick myosin filaments, became rearranged into a square lattice by the time they reached and connected to the dense Z-band (in mid-I-band) that divides one sarcomere from the next. Convincing Dr Bennett of this unlikely transition, before he left Seattle for the University of Chicago, required me to get more and better EMs. My final success in overturning his initial misgivings, and winning a senior class Honors Thesis Research award with my MD, gave me an early blessing of confidence that my way of making and analysing observations might fit me to include research in cell biology as part of my future career. Why the lattice switch from hexagonal to tetragonal, twice in every sarcomere? I don’t know — to me it’s still an answer looking for a compelling question. But the distinctive basketweave pattern of the Z-band, as seen in transverse sections, embodied a specific sense of twist, a possible clue that left-handed thin filament torque (arising from partial activation of resting muscle by the OsO4 fixative) might tend to straighten the inherent curvature of the four Z-bridges that connect each thin filament to four such filaments of opposite polarity that emerge from the opposite side of the Z-band. Thus, pursuing the possibility of filament torque expression in the Z-band as a component of force generation in striated muscle was the general goal I proposed to explore in applying, with Hugh’s encouragement, for the NIH post-doctoral funding that brought me in September 1963 to the new lab he’d left University College London to occupy at the MRC-LMB in Cambridge, with my wife Deirdre and son Tad as accompanying family.

Figure 7.2. Mike and Deirdre Reedy, dressed for St John’s May Ball, c.1965.

The peak moment of transatlantic travel stress for me came just after disembarking about 8.00 am at Southampton after the ocean trip from New York, and finding it all but impossible to understand my native language, as pronounced at the railway ticket window by an agent whose whim utterly controlled my chances of getting the family and me to Cambridge rather than Liverpool, Oxford or Edinburgh. Nevertheless, the tickets he issued clearly stated Cambridge, so my pulse rate came down as the train’s speed increased. Soon enough in Cambridge we’d rented a semi-heated house a convenient half-mile from the lab along Queen Edith’s Way, we’d survived a week of automobile touring to Canterbury, Beachy Head and Stonehenge as I slowly eased the arm and shoulder soreness I’d initially suffered from bracing myself against reverting to potentially fatal driving habits learned in America on the right (now suddenly wrong) side of the road, we’d learned the joys of getting around to Cambridge shops on bicycles and double-decker buses, and we’d become fondly familiar with the Beatles and the Stones on TV, with the confusingly long hair on young men and the distracting miniskirts and knee-high boots on young ladies, sights that we soon stopped noticing as my own hair grew long and Deirdre’s boot-tops and hemlines rose.

Hugh was away for our first two weeks in England, but a day or two after returning from our automobile tour Max Perutz, co-director with John Kendrew of the LMB, came into the lab, introduced himself as ‘Max’ and asked if I’d be so kind as to contact Moss Bros about fitting a rental tuxedo so I could serve as one of the ushers for his upcoming London lecture at the Royal Institution. I was flattered enough to pay my own tux rental and train fare to London, never to ask about reimbursement, and to conduct myself with the kindly formality I presumed essential to my high level of honorable service. I was especially happy to feel on comfortable terms with Max for all the years that followed. One very fine teatime in my first autumn, with late sunshine angling through rising smoke from a nearby cornfield burning off after-harvest stubble, I was pleased to spot Hugh with Max sharing their tea on an outdoor balcony extension of the third-floor lunchroom, chatting with a pleasantly mustachioed 70-ish gentleman in a tweed jacket, whose name I didn’t catch when Max introduced us as I sat with my tea to join them. The pleasure of the sun’s warmth and light slowed conversation to such a degree that I recall none of it, but after we broke up, I asked Hugh to tell me again the older man’s name. ‘Oh, that was Sir Lawrence Bragg.’ I was staggered to hear this, to have had tea unawares with a man so utterly legendary I’d only ever associated his name with the photo of him in my undergraduate physics textbook, sans moustache at age 25 when he’d shared the 1915 Physics Nobel Prize with his father for pioneering development of X-ray diffraction crystallography. Of course Max and Hugh had known Bragg for decades; he had founded the downtown lab where both began their work.

I came to the lab so primed to start trying my pent-up ideas for Z-band experiments on live frog sartorius and glycerinated rabbit psoas that Hugh chose not to suggest anything additional at the start – or even during our later progress reviews and discussions, because I continued to have more ideas and opportunities than I could finish testing in the lab. I think this accounts for the regrettable fact that we never developed any collaboration between the two of us in those days. We do now have an idea at last for some joint work in the next few years.

Five notable interactions with Hugh come to mind from my first three months in the lab.

I had no sooner settled in at the lab than I decided to cloister myself at home for whatever time it might take to finish writing up my Z-band discovery, by now partly scooped in a paper from Knappeis and Carlson. Perfectionism and inexperience slowed me to a wretchedly glacial pace. After about two weeks Hugh phoned my home and rather sternly asked whether and when I was planning to come into the lab. I squirmed for a couple of minutes, trying to explain my plan for the paper. Unconvinced, he simply said ‘I think you’d better get into the lab from now on’ and so I did; that paper was never finished apart from a brief note about the left-hand Z-twist in a Royal Society conference note I prepared a few months later.

Another lesser embarrassment came in the following month, when he’d given me use of the first diamond knife I ever handled for cutting thin sections; I’d only used glass knives before. We were cutting sections side by side, and it seemed to be taking me forever to face off the block down to the level where I’d be sectioning embedded muscle fibers, so I began to crank the advance of the Porter-Blum UltraMicrotome MT-1 faster and faster, telling myself: ‘Diamonds are very hard. Glass knives wouldn’t stand this for very long at all without dulling the edge.’ My 3–5 Hz cutting cycles were easily audible, but Hugh did not look up or comment. Later, at tea with him, I realised I’d not heard a similar sound of rapid cutting cycles from his microtome, so I asked him, ‘With a diamond knife, do you cut away the empty epoxy resin much faster when you’re facing the block to get down to the muscle, and then slow down to cut the sections for the EM?’ He said, ‘Is that what you were doing?’ I admitted as much, and asked, ‘Would you do that with a glass knife?’ I think he shuddered. I was afraid to meet his eyes. Speaking slowly in a low voice that captured 100 per cent of my attention, Hugh said, ‘Never! Not with a glass knife and not with a diamond!’ I muttered, ‘Oh lord, I may have ruined that part of the diamond, maybe 30 per cent of the edge length.’ He did not instantly banish me, though perhaps throttling an impulse to do so, but said instead, ‘Do try that edge again, then move sideways to cut from another part of the same edge to see if it’s any different. That knife has already been used a lot.’ And he said, ‘We’re getting a couple of new diamond knives in a few days. You can have one of those to take care of.’ After that, ‘Taking Care of a Diamond Knife’s Edge’ became more or less my first, middle and last names forevermore. And I soon developed a way to embed muscle fibers extremely close to the flat planar surface of the cured epoxy block, so I only had about 5 µm of empty epoxy to slice away before the knife began cutting longitudinal sections of muscle.

At quite the opposite pole of delight were the biweekly or monthly ‘coffee, biscuits and hi-fi musical evenings’ that Deirdre and I attended, with between four and eight other guests, in Hugh’s splendidly lovely second-storey rooms on the Cam river in King’s College for the duration of his year’s Fellowship there. I remember Audrey Glauert, Odile Crick, the Frank Stahls and Hugh’s recently widowed and kindly father, a retired postal inspector from Wales, among some of the guests. I particularly recall the supreme Scarlatti, but also remember my wife Deirdre’s happy triumph when she dared bring along Jazz Samba, a lovely bossa nova LP by Stan Getz and Charlie Byrd, as an alternative to the lively but totally classical music on which Hugh had been focusing his excellent hi-fi system. He tried Jazz Samba, and suddenly he was smiling and dancing himself around and across the room; he liked it! And I think Deirdre stood and danced with him a while, but I was not yet relaxed enough about dancing to join in. (Deirdre did finally teach me to jitterbug a few months later in the swaying bar-room car of an all-night ski train rocketing madly to Austria.) I was told by a friend of his about Hugh’s efforts to quit smoking while he still lived in those lovely King’s College rooms; he would resolutely pitch his last half-pack of cigarettes out the window into the winter night, but sometimes on finding next morning that the river was still frozen, he would slip down to the river bank and find a stick or a rake to help fetch back the suddenly precious discards.

I don’t mean this to sound at all silly. I learned how deeply addictive were my smoking habits from the foggy-minded need for nicotine that blurred those Sunday lab afternoons when I’d run short and the only open tobacco shop was too long a bike ride across town. It took me more than three years of pondering this discovery to quit, suddenly and forever, in early 1967 after I’d returned to the US and taken up a job offer with Wilfried Mommaerts at UCLA.

Once I knew Hugh well enough, I brought up the fact that he did not match my memory of his looks at his first 1957 seminar in Seattle. ‘Were you ever plump?’ I asked. ‘I could hardly recognise you, so fit and youthful, when we first talked in Seattle after your 1962 seminar. Had you lost weight?’ ‘Oh, I did put on a few extra pounds at first in America, but once I changed my diet I easily took them off again.’ ‘How?’ I had to ask. ‘Well, I gave up mayonnaise and potatoes. Nevermore! That’s all it took!’ It’s a secret I’ve never released until now.

Toward the end of November 1963, I went to a high-level meeting on muscle in London, sponsored by the Royal Society and organised by the two unrelated Huxleys, Hugh E. and now-deceased Andrew F., both noted for important work on muscle. Memory lights the opening day with fireworks from two events, exposure of my Z-band discovery to a fellowship who could judge its real value, and hearing at dinner of President Kennedy’s death. Lord Florey, the Society’s president, opened by congratulating A.F. Huxley, as ‘one of our members who has recently had some good news from Stockholm’ (the Physiology Nobel shared with Hodgkin and Eccles) to which those assembled grumbled ‘Hear hear!’ and offered a very short round of muffled applause. Later, I got to speak for three minutes just before lunch (or perhaps coffee?) about the left-handed sense of twist I’d discovered in the Z-band, when Jean Hanson kindly called me forward to offer a couple of slides in response to a question from the audience about the screw sense of the actin helix that she and Jack Lowy had recently imaged by negative stain EM. That evening’s party at a flat Hugh still kept in London was preceded by a wherever-you-like dinner hour in London. Arthur Rowe, the other Huxley lab postdoc, went with me to an Italian ristorante in Soho, where I all but laughed off a waiter who came out about 6.30 pm to tell us in broken English that ‘your President has been shot.’ I took him seriously 15–20 minutes later when he came out again and said, ‘your President has died now.’ When we got to the party, I quickly located Hugh to ask if he’d heard the news. He said ‘Yes, we’ve lost one of the good guys. Now there’s only the one!’ ‘Who do you mean?’ I asked. ‘I mean the Soviet premier, Khrushchev.’ ‘How do you mean that?’ And Hugh said, ‘It took both of them together to settle the Cuban missile crisis without getting the world into a nuclear war. Now only one is left.’ I held to that view when I saw Khrushchev in person during the 1964 international EM meeting in Prague; he was orating to a crowded plaza from a second-storey balcony, and I sent a short prayer of thanks to him as the good guy Hugh had made me aware of. Hugh’s politics were quiet, but within the next year, I had fully bought into his scepticism about the military urgency the US stated as necessary to stop a flood-tide of world Communism from spreading beyond Vietnam.

A year into my Z-band research, I and others had found a different Z-lattice pattern in relaxed muscle that became known as ‘small-square’, in contrast to the basketweave pattern I’d seen at first. It seemed to me that the transition from small-square to basket-weave could be effected by a thin filament rotation that could stretch and partly unbend the tight 90° kink in the Z-bridges that produces the small-square pattern. I began taking great pains to overcome the hysteresis of Siemens electron lenses so I could calibrate the magnification of Hugh’s Elmiskop 1a EM to within 2 per cent to provide an exact measure of Z-lattice spacing. This was true drudgery, but made interesting by my hope of success at a time when 10 per cent or greater error in EM magnification was often accepted as unavoidable. But just as I was about to vanish completely into this Z-band-motivated task, Ken Holmes and Oxford visitor Richard Tregear described to me some hard-to-explain changes they’d produced in the X-ray fiber diffraction pattern of insect flight muscle (IFM) from giant waterbugs when they transformed it from the rigor state, where Mg•ATP is absent, to the relaxed state by adding Mg•ATP. I offered to compare thin sections of the two states by EM, if we could first show that fixing IFM in the absence and presence of Mg-ATP preserved the diffraction pattern signatures of the two states. The answer to this was happily positive, as we found by X-ray diffraction of embedded muscle bundles that had been fixed in one state or the other before embedding in epoxy resin, and by optical diffraction or computed power spectra of EM images from longitudinal sections. Within about ten weeks we had our 1965 Nature paper in the press, showing that ATP-relaxed myosin crossbridges project at 90° to the thick filament axis, whereas rigor crossbridges form chevrons in which the myosin heads attach to actin at ~45° to the fiber axis, suggesting the end of a crossbridge power stroke. The methods I’d been developing for the Z-band work, especially for cutting and Mn-Pb staining of 20 nm thin sections, and the practices (like Z-control of vertical position, lens-current cycling to normalise hysteresis) that could hold EM magnification error to within 2 per cent, along with laser-based optical diffractometry of EMs introduced at LMB by Aaron Klug and Jack Berger, turned out to be precisely what was needed for the EM work partly described in my single-author paper of 1968. The Z-band problem was picked up in the 1980s by Margaret Ann Goldstein, and later brought back to my lab when her student, Robert Jon (‘RJ’) Edwards (Perz-Edwards = married name), made it the subject of his 1998 PhD with me at Duke University. This work, published in 2011, identified several procedures that can force the transition between small-square and basket-weave Z-band patterns.

Insect flight muscle subsequently became my main focus after 1965 and my exclusive focus after 1980 (apart from RJ’s Z-band work). It continues to be wonderfully informative. Hugh Huxley and Oxford’s J.W.S. Pringle (Tregear’s supervisor) generously set a precedent at the outset by letting Holmes, Tregear and I publish the initial structural work as sole authors in 1965, and we three each continued work on IFM in our separate and at times collaborative ways after that. I felt honoured and grateful beyond measure when, shortly before I left the LMB to return to America, Hugh invited me to consider whether I and my family might be willing to return to Cambridge within the next year or two to let me take up a staff position at the LMB. This appealed enormously to me, because Hugh Huxley’s presence there offered the best colleague I could imagine anywhere, while making the LMB a Grand Central crossroads for every muscle researcher on Earth, and the incredible support facilities (‘bottomless’ supplies counter, magnificent machine shop) meant one could get on with research itself rather than struggle with research infrastructure as I found I had to do in setting up a new EM lab in physiology at UCLA. On the day I finally did leave, rushing to Seattle to be present for daughter Heather’s birth, I went in to say goodbye to Hugh, and told him how wonderfully honoured and lucky I felt to have been able to work with him and around him. He said: ‘Those are the very words I wanted to say to you, Mike.’ Returning to make a life in Cambridge did not appeal much to Deirdre when I told her, so it never happened. But Hugh’s warm words that day 46 years ago gave me wings I can still fly on from time to time.

I’m running short of time. I wanted to say how Hugh’s contact with his longtime friends David and Dodie Robertson paved my way to Duke in 1969, how my LMB contact with Ken Holmes enabled the joys of my and Mary’s 1978–9 sabbatical in Heidelberg and the start of my increasingly helpful access to synchrotron X-ray diffraction for my muscle research, how Ken Taylor came to Duke from the LMB to build the last 30 years of collaboration with Mary and me on 3D EM structure of IFM, now growing to become 4D electron tomography of muscles freeze-trapped at strategically critical milliseconds, and especially more about my late wife and 35-year scientific partner, Mary, who took over so much that I could only do half-well and made it happen at the 100 per cent level. But I can’t possibly do justice to all the years and people who have mattered since I left the LMB, so here is where I must stop and give thanks to all those I’ve mentioned and what feels like hundreds more.

CHAPTER 8

The Medical Research Council Laboratory of Molecular Biology, January to July 1964

Ignacio Tinoco, Jr.

Department of Chemistry, University of California, Berkeley, CA

Figure 8.1. Ignacio Tinoco in 1964 and 2010.

My wife, Joan, nine-year-old daughter, Kathy, and I arrived in our wrong-side-drive VW bug in Cambridge on 2 January 1964. I was to spend six months at the MRC Molecular Biology Laboratory as a sabbatical visitor from the University of California, Berkeley. We found a modern house to rent on Fendon Close, right across Hills Road from the lab. Modern meant that there were gas pokers to light the coal to heat the hot water and the house. We quickly learned that the warmest places in Cambridge were our car and the tropical rainforest building in the University Botanic Garden. Of course, we found many differences from the US. I could understand why the wine store wouldn’t sell me a 1920s claret because they were convinced I wouldn’t know how to decant it and would probably bring it back. But I was annoyed when I tried to buy a book for my daughter at Heffers. They asked her age, then told me she wasn’t old enough for that book and refused to sell it to me.

My life at the LMB was also very different and very pleasantly surprising. I had applied to John Kendrew for a desk in the lab and I knew I was very lucky to be accepted. It was a year after four Nobel Prizes were awarded to the lab, so everybody wanted to work there. My roommate was David Davies, and although he was a protein enthusiast and I favoured nucleic acids, particularly RNA, we had a lot to talk about, such as tennis. I played for the first time in my life on grass courts. One day at the lab someone mentioned that they didn’t know that I was a member of King’s College. When I told them I wasn’t, they asked how come I was playing on their courts. I’m sure David knew it wasn’t proper for non-members to use College courts, but also knew that nobody would question us.

My interests when I came to Cambridge were on the optical properties of nucleic acids and proteins. The molar extinction coefficient of DNA is 30 per cent lower for the native duplex compared to the denatured molecule. The cause of the hypochromicity was puzzling. Polypeptides underwent similar changes in UV absorption following a coil-to-helix transition. Even more striking effects occurred in the circular dichroism of polypeptides and polynucleotides on helix-coil and native-denatured transitions. I was trying to relate the spectroscopic changes to the interactions between the nucleic acid bases, or the amides, and thus to the 3D structures of the molecules. The only person interested in this subject at the LMB, as far as I could tell, was Aaron Klug. I didn’t get specific advice from him or anybody else, but the overall atmosphere at the lab was very helpful to my research at that time and, as it turned out, for the rest of my career. What was so special about the environment that encouraged science? Were the people working there uniquely talented and thus able to make groundbreaking discoveries? Or were they average scientists stimulated by the ambience to work hard and think even harder, to accomplish something new? I am convinced that the scientific discoveries at the lab were due to the interactions among the whole group; the results were much more than the sum of their parts.

At coffee in the morning, lunch at noon, and tea in the afternoon there were always discussions about what experiments to do, and how to interpret them. Whatever the conversation, having a Nobel Prize-winner at the same table for tea, ready to discuss problems, allowed us to recognise that they weren’t superhuman, but were more or less normal people. Crick always wanted experiments that gave clear-cut, yes-or-no answers. A quantitative measurement of the change in circular dichroism spectrum when DNA switched from a right-handed helix to a left-handed helix was much less interesting than an experiment that immediately told you the DNA was left-handed, not right. Sanger’s group was starting DNA sequencing, and the problem I remember them discussing was where to borrow a sewing machine. They were doing paper chromatography and wanted to sew one sheet onto another to transfer the lane separated by one solvent to a new sheet for a second separation. Obviously all sorts of problems were discussed, some profound but most trivial. However, what came through in the conversations was that everybody was excited about their work and wanted to share their excitement, or their frustrations. There did not seem to be fear that anyone ‘would steal their idea’, or that somebody else would point out how stupid they were for not knowing an obvious fact. You would be told that a critical control had been omitted, or that a more clear-cut result would result if the experiment were done differently, but it was clear that the goal was to produce better science, not to put you down. The attitude in the LMB seemed to be: it is us against the world. We compete strongly with the world (and usually win), but within the lab we only collaborate.

I have highlighted the scientific discussions that occurred in the cafeteria, but of course there were many other topics of interest at the time. In 1964 the Beatles easily conquered the US after concerts in New York and the capital, while the US was beginning its own war in Vietnam. One topic that I don’t remember being discussed was money. Nobody seemed to worry about funding for chemicals, for stipends for technicians, staff and students. Apparently, the necessary money appeared magically.

I am sure my memory of the LMB filtered through nearly 50 years of other experiences is very different from the reality. However it was clear then, and it is even clearer now, that it was a very special place. People were friendly, welcoming and helpful; Leslie Barnett was especially kind to my family and me. The research facilities were excellent. My main research tool was the computer. Young women would take suitcases of punched cards by train to London twice a day and return with printed sheets of output. The turnaround time of less than 24 hours was impressive. I was never asked what I was calculating or why. At Berkeley I had access to computers, but I had to pay for every minute through my funding from the NIH and the Atomic Energy Commission (which eventually became the Department of Energy). Maybe the line at the top of each output telling me how much time I had used, and how much time I had left in my account, was a sufficient distraction to make me miss an important discovery in the data. Maybe the reminder of the cost discouraged experiments with high probability of failure, but with important consequences if successful.

My memories of the lab meld into my ideas of the perfect place to do research. The building is designed to stimulate interactions among the research groups with convenient gathering places to meet. The researchers have the latest instrumentation maintained by engineers for optimal performance. There are experienced specialists to do routine preparations and purifications of biological materials, and competent technicians to help with novel methods. The infrastructure of the lab including mechanical shops, analytical facilities, purchasing, food service and maintenance, works effectively. All this is orchestrated by a manager who acquired the money, hired the people, dealt with any friction among the staff, and made sure everything ran smoothly and efficiently. This all-wise manager would also know which researchers to encourage and to keep at the LMB, and which to help get other positions more suited to their talents. There would be collaboration among the scientists, who would meet every day at the cafeteria, but nobody would tell them what experiments to do or not do.

I think the most difficult task in establishing this lab in Camelot is to pick the people. This is a continuous job because if mistakes are made they must be rectified immediately, and the scientists who lose their interest and enthusiasm must be replaced. Assuming that the people working in the lab are not crazy, I think the criterion for support should be dedication to their science. People who are excited about what they are doing, who know and care about what is going on in their field, and who are always thinking of problems to solve will do good science that deserves support. People who find their hobbies more interesting than science, or who continue making small increments to a project finished years ago, deserve a change in their position.

My six months in Cambridge continue to make a difference in my scientific life. Before my research interests evolved away from ultraviolet absorption, optical rotatory dispersion (ORD) and circular dichroism (CD) of nucleic acids and proteins, my students used CD to show that double-stranded RNA could also become left-handed, although at much higher salt concentrations than for DNA. Most people were not impressed. I remember before my seminar at a Midwestern university a faculty member said he was eager to hear what I had to say about Z-DNA. When I told him I was talking about Z-RNA, he left. When I was asked what was the biological significance of Z-RNA, my usual answer to the audience was that it was of interest to a human. We added other optical methods to our repertoire: the differential scattering of right and left circularly polarised light (circular intensity of differential scattering, CIDS), and fluorescence-detected circular dichroism, FDCD. CIDS was used to determine the helical parameters of the sperm from the Mediterranean octopus Eledone cirrhosa, and FDCD characterised the conformation around the fluorescent base in the anticodon loop of yeast phenylalanine transfer RNA.

The thermodynamics and kinetics of double strand formation in RNA continue to be a long-term interest; we want to be able to predict whether a secondary structure forms and what its melting temperature will be. Our first effort, published in Nature in 1971, used ‘stability numbers’ instead of free energies to characterise a possible base-paired structure. We reasoned that biochemists (molecular biologists had not been invented yet) would not understand that negative free energies meant more stable structures. We thus chose a +1 stability number for each A•U base pair and a +2 for each G•C. Loops and bulges were given negative stability integers. In our next publication we switched from just counting base pairs, to considering nearest-neighbour sequences of base pairs, and also reported stabilities as Gibbs free energies relative to the single strand. Since then experimental and theoretical work by many scientists has brought the goal of prediction of RNA and DNA stability much closer to fruition.

In the 1970s NMR magnets were becoming strong enough to make studies of small oligonucleotides useful. We started with a 200 MHz instrument, graduated to a 360 MHz, then to a 600 MHz. We looked first at dinucleoside phosphates, our samples for the UV absorption spectra we were trying to understand at Cambridge. With increasing resolution and 2D spectra we moved up to RNA solution structures of extra-stable tetraloops, pseudoknots, kissing hairpins and ribozymes. I am pleased that two students working with me in the 1980s spent time at the LMB in Cambridge. Gabriele Varani, a postdoc in my lab, was there as a senior scientist from 1992–2001; Fareed Aboul-ela, a graduate student, was a postdoc in Cambridge in 1994–7. I was happy to hear from them that some things have not changed. Money seems to appear magically, scientists are given freedom to follow their own instincts in research, and there are wide-ranging discussions of all topics in the cafeteria.

During the last ten years my research group has switched completely to single-molecule studies. The idea that a single molecule in a solution or in a biological cell could be followed and characterised would have been branded science fiction in the 1960s. In 2000 nearly any single-molecule experiment was considered novel and worthy of Science or Nature. Now single-molecule experiments are routine enough that for the most part they are judged by the results produced. We have used laser tweezers to study translation one ribosome at a time. Beads are attached to the ends of a messenger RNA in the form of a base-paired hairpin. One bead is held on a micropipette, the other is in a laser trap. As a ribosome translates the hairpin, double-stranded RNA becomes single-stranded and the end-to-end distance between the beads increases at constant force. We found that translation takes place by a step-pause-step trajectory. Each step is the translocation of the ribosome by three nucleotides, one codon. Each pause occurs while the next amino acid is brought in to the ribosome and the new peptide bond is formed. We are also studying the folding of the nascent protein on the surface of the ribosome. Instead of using heat or 6 M urea to unfold the protein, we pulled on its ends. The ribosome acts as a chaperone on the nascent protein; it slows its folding and prevents misfolding.

I still try to continue the environment that I learned at the LMB where the researchers in my group don’t compete, but collaborate. I observe unhappily that in top-ranked American universities students and postdocs, even in the same group, compete with each other. A student is afraid to mention an idea for fear that it will be presented to the professor by a fellow student as their idea. Everybody is too busy to help a newcomer. Everybody is too busy to have coffee or lunch together, and nobody drinks tea. The professor has lunch in the office while writing grants, and the student is lucky to talk to him or her once a week. Scientific ability is measured quantitatively by the amount of funding obtained, and by first-or-last-author papers in Nature and Science. To publish in those venues the spin in the paper is the most important. The first hurdle to get over is to have the paper sent out for review. I was given the advice that as the editors only read the abstract, you should claim a cure for cancer or equivalent there. The referees may find the science acceptable after a few more experiments, as long as the offensive claim in the abstract is removed. The ultimate measure of your contribution to science is your Hirsch Index of cited publications. Whether your most highly-cited paper is wrong is irrelevant.

New fundamental discoveries continue to be made in laboratories all over the world, and of course they will continue to be made. We will never reach the end of learning in any area, particularly science. The rate of progress is accelerating in acquiring new data, but to know whether our understanding is also accelerating, we must wait for the next 50 years.

CHAPTER 9

More than a Stopover: Eight Months with Sydney Brenner (1964)

Jonathan Beckwith

Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA

Figure 9.1. Jon Beckwith, Roger Freedman and Tony Stretton, on the canteen balcony at LMB. Photo by Mark Bretscher.

My five peripatetic years of postdoctoral work included an unanticipated and impactful period in Sydney Brenner’s lab. After beginning my first postdoctoral year on the west coast of the United States with Arthur Pardee in 1961 at the University of California in Berkeley, I moved continually eastward: to New Jersey when Art decided to take a position at Princeton University; to London and Bill Hayes’ group at Hammersmith Hospital in 1963; to the LMB in Cambridge, England with Sydney in 1964; and finishing off with a year at the Institut Pasteur in Paris with François Jacob. This extended five-year post-PhD period was driven by my determination to land a spot in François Jacob’s lab.

In 1957, I started as a PhD student in the chemistry department at Harvard University. However, during my graduate period I slowly transferred my allegiance to biochemistry and then to bacterial genetics. Both of these latter fields seemed to be at a very exciting stage at that point as they moved towards a fusion into the newly named field of molecular biology. I was particularly influenced by a microbiology course I took, taught by Bill Sistrom and Jim Watson. In a paper I wrote for the course on permeases, I was led to the work of Jacques Monod, François Jacob, Élie Wollman, George Cohen and their collaborators. I was seduced by the clean logic that guided their experiments, the ingenuity of their genetic approaches, and the elegant French writing style characterised by a surprising economy of words.

I had first applied to Jacob while in my last year of graduate school. He suggested that I needed to gain more experience in genetics first and suggested that I train with Art Pardee. I followed Jacob’s advice and spent two years of postdoctoral stay with Art. Art had initiated a study of a class of mutations (lacO0) that inactivated the Lac operon, originally isolated by Jacob, Monod and their coworkers.1 He asked me to take over the project. The O0 mutations were point mutations that mapped early in the lac operon and resulted in the failure to express all three genes of the operon, lacZ, lacY and lacA. Jacob, Monod and coworkers had proposed that these mutations were in the Lac operator, a site that they had previously shown to be the site of repressor action. They now posited that the repressor and transcription machinery competed for the site where expression of the operon was initiated. The operator was also, in this model, what we would call today the promoter. Based on this attractive and parsimonious model, Art proposed to use properties of revertants of the mutations that restored operon expression to provide an explanation for how different genes come to be expressed at different rates. He thought that it might be possible to isolate O0 revertants that behaved like wild-type as well as those that expressed the operon at intermediate levels. If revertants with various levels of expression could be obtained, it would be consistent with the idea that such a site could be mutationally altered to give different degrees of operon functioning. This explanation for differing levels of gene expression may seem self-evident today, since we have learned so much about promoters and what determines how active they are in permitting transcription initiation. However, at that time, no one was even considering the question.

Art handed over the project to me and we showed that revertants could be obtained that showed a wide range of amounts of operon expression, consistent with his idea.2,3 I then began to genetically analyse the revertants he had obtained. While trying to map the mutations in these revertants to expected locations in the Lac operon, I found to my surprise that the mutations restoring Lac operon function to intermediate levels were due to suppressor mutations unlinked to Lac. Since, at the time, it was hard to believe (I am not sure why) that such suppressors could be acting at the transcription level, we began to question the Pasteur scientists’ interpretation of these mutations.4

I accomplished much of this work when I was nearing completion of my second year of a three-year NIH fellowship in Art’s lab. Having by that time learned at least some bacterial genetics as Jacob had suggested to me, I again wrote to François and asked him whether he now had an opening for me in his lab. He replied this time that his lab was full at the moment and he would not have a free space in his lab for a while. However, as I was still determined to get broader experience in bacterial genetics, I sought and obtained a place in the Medical Research Council’s Microbial Genetics Research Unit at Hammersmith Hospital in London, headed by Bill Hayes. Hayes was famous for his work that determined the nature of many features of bacterial conjugation including the presence of the F factor (today called the F plasmid) in E. coli donor cells. Perhaps now, separated from Paris and the Pasteur Institute by only the English Channel, I would be able to visit François and convince him of my worthiness.

In Bill Hayes’ unit I was able to continue on an aspect of the lacO0 project that I had also begun in Art Pardee’s lab. This was based on an idea suggested by a graduate student in Art’s lab, John Gerhart. He pointed out that it might be possible to select for revertants of the lacO0 mutant that only restored expression of the second gene of the Lac operon, lacY. The lacY gene encoded the lactose permease, a membrane transport protein for lactose that also transported the sugar melibiose, a sugar that was hydrolysed by a different galactosidase than that encoded by the lacZ gene. If I selected for growth on melibiose, I might obtain revertants that restored expression of lacY but not lacZ. They would be Mel+ but not Lac+. I have no memory today of what I hoped to gain from doing such a genetic selection, but I went ahead with it. Most of the revertants that grew on melibiose but not lactose as a carbon source were due to deletions of the region in which the Oo mutations mapped.5 This collection of deletions was quite important, as in conjunction with studies with Sydney Brenner on suppressors that I will describe below, it led to a revision of the interpretation of the Oo mutations. In addition to this work, I collaborated on a project on episome formation with two graduate students in Bill Hayes’ unit, John Scaife and Paul Broda.6

A few months into my stay in London, I was surprised to receive a call from Sydney Brenner who was then working at the Medical Research Council’s LMB unit in Cambridge. He asked if he could visit me in London to talk about an idea he had about the suppressors of the Oo mutations I had characterised in Pardee’s lab. He had read my paper on the suppressors and thought that they might represent a new class of suppressors of chain-terminating mutations different from the well-studied amber suppressors. The amber mutations had been characterised by studies on bacteriophage T4 and alkaline phosphatase and were thought to introduce protein chain-termination signals during the translation of proteins.7,8,9 Sydney suggested that the lacOo mutations I had worked with might have introduced a chain-terminating signal different from that which amber mutations introduced and that the suppressors I had detected restored translation to genes which had this alternative termination signal. He proposed that we collaborate on these suppressors and invited me to spend time in his lab when I finished the third and last year of my NIH postdoctoral fellowship. I handed him my E. coli strains harbouring the suppressor mutations so that he could start right away to test his hypothesis.

This invitation may well have rescued my scientific career. I was in the last year of my fellowship; the hoped-for postdoctoral position in Jacob’s lab had not materialised; and I had no prospects for a faculty job back in the States. My wife Barbara and I had been discussing the possibility that I might go back to school to train in another, non-scientific field. It had seemed, early in 1963, that my scientific career might come to an end. Nevertheless, on the basis of this invitation from Sydney, I tried once again for the postdoctoral position in Jacob’s lab, applied to the Jane Coffin Childs Foundation for postdoctoral support, and was interviewed for a faculty position at Harvard Medical School. Within a few months, I had a fellowship, an invitation from Jacob to do postdoctoral work in his lab, and a job offer from Harvard. Of course, these sudden successes weren’t only because of Sydney’s interest: my evidence that the Pasteur group may have been wrong about the Oo mutations thrilled some scientists, who, I suspect, gloated over the fact that these highly successful people could make mistakes. I remember Boris Ephrussi saying to me in this regard: ‘I never did like big operators!’ Perhaps partly for this reason, my work was becoming known, even though it refuted a minor detail in the theory of gene regulation that the Pasteur group had developed.

Even before I moved to Cambridge at the beginning of 1964, Sydney had made considerable progress with his studies on the activity of my lacO0 suppressors in restoring function to bacteriophage T4rII chain-terminating mutants. His results showed that my suppressors did indeed suppress a new class of chain-terminating mutations distinct from the amber mutations. He named this new class ochre mutants.10 In the meantime, I had shown that deletions internal to the Lac operon that removed the region in which the Oo mutations were located restored functioning of the operon5 (even though the function of the lacZ gene was destroyed; see above). Thus, the fact that the suppressors of lacO0 mutations almost certainly acted at the level of translation and my own findings that the region where the lacO0 mutations mapped was not essential for expression of the Lac operon, made it seem highly unlikely that these mutations were defining the site of transcription initiation of the Lac operon. Instead, we concluded that the mutations were likely only extreme examples of polar mutations that were beginning to be found in many operons.10,11 By causing polypeptide chain termination, these mutations induce a downstream transcription termination process.12

I only worked for eight months in Sydney’s lab at the LMB, partly because I was still determined to fulfill my goal of being, if only for a year, a Pastorien and work in François Jacob’s lab. Yet the time I spent in Cambridge was, by far, the most intense and formative scientific experience of my years of training. It was not only the very bright postdoctoral fellows and students in Sydney’s lab – Roger Freedman, Tony Stretton, Ethan Signer, Anand Sarabhai, Bob Rownd, Sam Kaplan and David Zipser; nor the highly successful people who populated the LMB – Francis Crick (sharing the same room as Sydney), Fred Sanger, John Kendrew, Max Perutz, Aaron Klug, Leslie Orgel and John Smith and the discussions at tea in which many of them participated. The interactions with Sydney were by far the most influential of my stay in Cambridge. In the main, Ethan Signer and I worked on mapping the collection of amber and ochre suppressors, including those I had isolated.13 Sydney would often be first in the lab in the morning, and would have looked at my plates and already puzzled out the meaning of my experiments before I had arrived. We would talk frequently and for long periods, I mainly ‘sitting at his feet’ (figuratively) and, I am convinced, absorbing a way of thinking about science. One of the lab members told me that Sydney was impressed with me; but I thought at the time that it could only have been because I sat and listened to him for so long, perhaps with something that appeared to be a wise look on my face? I was being taught how to think and think quickly, as Sydney’s thoughts raced through so many ideas. There was always the hint of a smile on his face and his disquisitions were accompanied with a humorous undertone which did not reflect cynicism but rather an extreme pleasure at talking about new ideas and ways in which genetics would provide ways to explore them. I was privileged, at one of these sessions, to hear him talk about his new idea of studying neurobiology by mapping out the nervous system of nematodes. He had been thinking about this for a while, spending much of his time in the library reading up to find the most appropriate nematode to work with. While I worried that I wasn’t having much of a chance to think on my own during these conversations, I left Cambridge in August 1964 feeling that in eight months I had absorbed a way of doing science that became my way.

I can think of two major ways in which the collaboration between Sydney and me influenced work that we each took up in our own labs. At a time in which the genetic code was beginning to be deciphered, Sydney came up with an ingenious approach to determine the nature of the presumed codons that represented the amber and the ochre chain-termination signals. Using information on the nature of some of the already deciphered codons for amino acids, information on what codons were connected to the amber and ochre codons from mutational data, and studies on the mutagens that would induce amber and ochre mutations or revert them, Brenner, Stretton and Kaplan deduced that the codons for the two were UAG and UAA, respectively.14 This is one of the most beautiful papers from a period in which so many profound and foundational results were appearing. It is a classic example of the power of bacterial (and bacteriophage) genetics that allows one to conclude a very biochemical fact ‘without doing the biochemistry’ as Sydney had put it.

The second consequence of the findings from Sydney’s and my experiments was that they raised for me the following question: if the operator is not the site of transcription initiation, where is transcription of the Lac operon initiated? By the time this question was being posed, Jacob and coworkers had come up with a name for such a site – the promoter. They initially proposed, based on indirect evidence, that the promoter was between the operator and the lacZ gene.15 The following work I describe showed that this conclusion was incorrect. Having finished my nearly five years of postdoctoral work, I took a position in the department of bacteriology and immunology at Harvard Medical School. With John Scaife arriving in my lab as a postdoctoral fellow, we immediately set out to isolate and map promoter mutants. We devised a way to screen for mutants that were leaky for lactose metabolism on certain lactose-containing indicator agar. We did indeed obtain such leaky mutants which expressed all three genes of the Lac operon at a low level and were, therefore, candidates for promoter mutants.16 (In this way, we avoided picking up the strong polar mutations that had earlier muddied the waters.) These leaky mutations all mapped upstream from the Lac operator instead of between the operator and the lacZ gene.17,18 When we later detected the transcriptional activator CRP (cyclic-AMP binding protein) that was required for Lac operon expression, we showed that some of the ‘promoter’ mutations reduced affinity of the transcriptional activator (CRP) for the region adjacent to the promoter.19,20 Other mutations that directly altered the promoter itself mapped between the CRP-binding site and the operator.

Finally, in a collaboration between Sydney, David Zipser and Austin Newton, we showed that the polar mutants in the lacZ gene exhibited a gradient of polarity.22 Chain-terminating (or frameshift) mutations mapping early in the lacZ such as the formerly named O0 mutations almost completely eliminated downstream operon expression (e.g. that of lacY), while mutations later in the gene only partially reduced lacY expression. Such mutations near the end of the lacZ had minimal effects on downstream gene expression.

To some extent, when I set up my own lab at Harvard, I modelled it after Sydney’s. I tried to spend the lengths of time talking with the students and postdocs in my lab that Sydney had granted me. To their chagrin, I also used to go into the warm room and look at their plates before they had come into the lab. But, most importantly, I learned how deeply one can probe into biological problems by generating creative ways of manipulating cells genetically.

For seven or eight years after I arrived at Harvard, we worked on issues related to gene regulation that increasingly required the application of biochemical techniques for working out the molecular details. Since I was convinced that my skills were in developing ways to use genetics to solve complex problems, I decided to seek new problems to which the power of bacterial genetics could be applied. Inspired by my fellow faculty member Luigi Gorini, who was trying to understand the mechanism whereby bacterial cells can secrete proteins across the cytoplasmic membrane, I decided to see if we could use genetics to get some answers to this question. I felt that, as Jacob, Monod and coworkers had used genetics to open up the field of gene regulation, more complex problems of a cell biological nature could also be illuminated by genetics. Using this approach, we were successful in finding ways to obtain mutations that interfered with protein secretion and that revealed many of the components of the secretion machinery.23,24 We have continued in this genetic vein, providing genetic techniques for analysing membrane protein topology,25 discovering the enzymes involved in protein disulfide bond formation,26,27 and unravelling aspects of the cytoplasmic reducing environment, always with genetics as a means of opening up these studies.28,29 Certainly, the confidence that I had in this approach was ultimately inspired by the early work of Brenner, Crick, Jacob and Monod whose genetic experiments revealed basic biological mechanisms.

REFERENCES

1. F. Jacob, D. Perrin, C. Sanchez and J. Monod, 1960. The operon: a group of genes whose expression is coordinated by an operator. Comptes Rendus des Séances de l’Académie des Sciences 250, 1727–9.

2. A.B. Pardee and J.R. Beckwith, 1962. Genetic determination of constitutive enzyme levels. Biochimica et Biophysica Acta 60, 452– 4.

3. A.B. Pardee and J.R. Beckwith, September 1962. Control of constitutive enzyme synthesis. In ‘Informational Macromolecules’, Rutgers University, 255–69.

4. J.R. Beckwith, 1963. Restoration of operon activity by suppressors. Biochimica et Biophysica Acta 76, 162–4.

5. J.R. Beckwith, 1964. A deletion analysis of the lac operator region in E. coli. Journal of Molecular Biology 8, 427–30.

6. P. Broda, J.R. Beckwith and J. Scaife, 1964. The characterization of a new type of F-prime factor in E. coli Kl2. Genetical Research 5, 489–94.

7. R.H. Epstein, A. Bolle, C.M. Steinberg et al, 1963. Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symposia on Quantitative Biology 28, 375–96.

8. A. Garen and O. Siddiqi, 1962. Suppression of mutations in the alkaline phosphatase structural cistron of E. coli. Proceedings of the National Academy of Science of the United States of America 48, 1131–27.

9. S. Benzer and S.P. Champe, 1962. A change from nonsense to sense in the genetic code. Proceedings of the National Academy of Science of the United States of America 48, 1114–21.

10. S. Brenner and J.R. Beckwith, 1965. Ochre mutants, a new class of suppressible nonsense mutants. Journal of Molecular Biology 13, 629–37.

11. J.R. Beckwith, 1964. Restoration of operon activity by suppressors, in H. Stubbe (ed.), Structure and Function of the Genetic Material (ErwinBaurSymposium III, 1963). Abhdlg. d. Dadb., Klasse f. Medizin, Mo. 1, 119–24.

12. J.P. Richardson, C. Grimley and C. Lowery, 1975. Transcription termination factor rho activity is altered in Escherichia coli with suA gene mutations. Proceedings of the National Academy of Science of the United States of America 72, 1725–8.

13. E.R. Signer, J.R. Beckwith and S. Brenner, 1965. Mapping of suppressor loci in E. coli. Journal of Molecular Biology 14, 153–66.

14. S. Brenner, A.O. Stretton and S. Kaplan, 1965. Genetic code: the ‘nonsense’ triplets for chain termination and their suppression. Nature 206, 994–8.

15. F. Jacob, A. Ullman and J. Monod, 1964. The promotor, a genetic element necessary to the expression of an operon. Comptes rendus hebdomadaires des séances de l’Académie des sciences 258, 3125-8.

16. J.G. Scaife and J.R. Beckwith, 1966. Mutational alteration of the maximal level of lac operon expression. Cold Spring Harbor Symposia on Quantitative Biolology 31, 403–08.

17. K. Ippen, J.H. Miller, J.G. Scaife and J.R. Beckwith, 1968. New controlling element in the lac operon of E. coli. Nature 217, 825–7.

18. J.H. Miller, K. Ippen, J.G. Scaife and J.R. Beckwith. The promoteroperator region of the lac operon of E. coli. Journal of Molecular Biology 38, 413.

19. G. Zubay, D.O. Schwartz and J.R. Beckwith, 1970. The mechanism of activation of catabolitesensitive genes: a positive control system. Proceedings of the National Academy of Science of the United States of America 66, 104–110.

20. J.R. Beckwith, T. Grodzicker and R. Arditti, 1972. Evidence for two sites in the lac promoter region. Journal of Molecular Biology 69, 155–60.

21. J. Hopkins, 1974. A new class of promoter mutation in the lactose operon of Escherichia coli. Journal of Molecular Biology 87, 715–24.

22. A. Newton, J.R. Beckwith, D. Zipser and S. Brenner, 1965. Nonsense mutants and polarity in the lac operon of E. coli. Journal of Molecular Biology 14, 290–96.

23. D. Oliver and J.R. Beckwith, 1981. E. coli mutant pleiotropically defective in the secretion of E. coli proteins. Cell 25, 765–72.

24. P. Schatz and J. Beckwith, 1990. The genetics of protein secretion in Escherichia coli. Annual Review of Genetics 24, 215–48.

25. C. Manoil and J. Beckwith, 1986. A genetic approach to analyzing membrane protein topology. Science 233, 1403–08.

26. J.C.A. Bardwell, K. McGovern and J. Beckwith, 1991. Identification of a protein required for disulfide bond formation in vivo. Cell 67, 581–89.

27. H. Kadokura and J. Beckwith, 2010. Mechanisms of oxidative protein folding in the bacterial cell envelope. Antioxidants and Redox Signalling 13, 1231–46.

28. A. Derman, W. Prinz, D. Belin and J. Beckwith, 1993. Mutants that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262, 1744–46.

29. E.J. Stewart, F. Åslund and J. Beckwith, 1998. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. The EMBO Journal 17, 5543–50.

CHAPTER 10

Memories of the LMB (1964–5; 1977)

Stephen Harrison

Harvard Medical School, Boston, MA

I spent two intervals at the LMB – one in 1964–5 as a student; the other, in 1977, while on a one-semester research leave from Harvard. I arrived for the first time in September 1964, a very callow 21-year-old despite four years at Harvard, to be a visiting graduate student with Aaron Klug. I took the bus from town and walked up the hill to the laboratory – a single, very new building. The TMV model, a helical array of styrofoam loaves, in the glass-enclosed staircase made a vivid impression.

Aaron proposed that I work on efforts to reassemble turnip crinkle virus (TCV) in vitro, a project initiated by Reuben Leberman. Although the classic reassembly experiments with TMV were a decade old, no one had achieved a similar result with a spherical virus. In the course of the autumn, however, I decided that I would really like to learn crystallography. A series of lectures that Max had given at Harvard during the previous year had persuaded me to study structure; X-ray crystallography was clearly the way to do that, so I approached Aaron about a crystallographic project. He gave me a book to digest that began with goniometry and crystal form and then went on to symmetry of point groups and space groups – in short, an assignment intended either to discourage or to suggest that the subject was a daunting one – perhaps to keep me focused on the physical chemistry of virus assembly. I came back in a couple of weeks, said that I had finished the book, and asked what should I do next. I suppose my persistence with so dull an assignment was convincing. I took over the characterisation of TCV crystals that Bill Longley, about to leave for a postdoctoral stage with Don Caspar in Boston, had begun in his thesis.

Dealing with virus crystals was a curious way to learn X-ray crystallography. It took me two weeks to gain the dexterity needed to mount the extremely fragile TCV crystals in quartz capillaries. But a splendid consequence of needing a microfocus generator was to find myself next to Hugh Huxley (a luminary in my youthful pantheon because of the sliding filament model, which I had found inspiring in its elegance when I learned about it in the Woods Hole Physiology course). Hugh, along with Ken Holmes, was recording exquisite diffraction patterns from live muscle, using a specially built mirror-monochromator camera. Ken became a close friend; six years later, I spent a postdoctoral year with him in Heidelberg, just before starting a faculty position at Harvard. Another great advantage of working on a microfocus generator was that you were not at risk of being pre-empted by Max.

Max was the essential spirit of the place. While I had been introduced to him when he visited Harvard, I had no reason to suppose he knew who I was. Nonetheless, just a few weeks after I arrived, he came up to me in the canteen and asked how I was getting on. The gesture made a lasting impression. When Max wasn’t in a lab coat exposing and developing X-ray diffraction pictures, he was in the model room, working on a model of haemoglobin based on homology with myoglobin. The experimental high-resolution haemoglobin map was yet to come. Thirteen years later, when I built a Kendrew model of the tomato bushy stunt virus (TBSV) subunit, I realised what an exhausting process that must have been.

During that year, the genetic code was rapidly emerging from the experiments of Khorana and Nirenberg. Francis Crick and Sydney Brenner shared an office, and I have a vague and probably inaccurate memory of codons being added to a list on a blackboard as the news came in from abroad. Sydney, with Tony Stretton and others, worked out the amber and ochre stop codons at that time.

Some time in the winter, David Phillips and three coworkers came from London to report on the status of the lysozyme project. Their title was ‘Lysozyme: two steps forward, one step back’ – I don’t recall the specific nature of the step back, but it did not impede progress for very long. A memorable moment in the four-person seminar was when David introduced the last speaker, a student named ‘Miss Johnson’ (Louise, of course), who had crystallised an enzyme-substrate complex. Francis, hitherto somnolent in the front row, suddenly sat up and took notice. A few months later, Philips presented the completed lysozyme structure at a special, late-morning lecture at the Royal Institution. Laboratory members motored down to London (I managed to get a ride in the rear of the lab van; Max was in the front seat, and I think Paul Sigler was another of the passengers). After the lecture, in what I remember as a very grand and elegant hall, we were invited to inspect the wire model of lysozyme in an adjacent reception area. I was excited to see β-strands and disulfide bonds. While most of us stared at the model, Francis seemed more interested in ogling Miss Johnson. Uli Arndt, who had worked at the RI, gave me a tour, showing me the famous hydraulic lift.

In early June, I joined Gwynne Goaman, Joyce Cox (later Baldwin), and John Baldwin in an excursion to Glyndebourne. The opera itself (Cimarosa’s Il matrimonio segreto) was enjoyable enough, but less memorable than the occasion itself. We changed into evening clothes en route (a carefully selected petrol station, I think). A high point, as we finished our picnic on the lawn during the interval, was to encounter John Kendrew, in the company of a lady scientist I knew from Boston.

During that year, Aaron was developing his approach to the analysis of electron micrographs. He had realised that images from negatively stained particles were often projections of two-sided images, although one-sided contrast was occasionally evident. For periodic arrays (helical assemblies or two-dimensional lattices), he was using optical diffraction to separate the contributions from top and bottom. Jack Berger was commuting to Manchester to use the optical diffractometer in Lipson’s laboratory, until a similar instrument was installed at LMB in a shaft near the staircase. For icosahedral viruses, the initial analyses were based on calculated projections of various simple, ‘capsomere’ models. For the work on human wart virus, Aaron explained to me that it helped to name the characteristic views, as a mnemonic: ‘owl’s eyes’, ‘threefold terraced view’, and the like. Don Caspar came for the summer of 1965 and decided to create a macroscopic version of negative staining, by encasing models of icosahedral viruses in plaster of Paris and recording medical X-ray films using a facility in the radiology department that shared the building with LMB.

My TCV project moved forward slowly but steadily. I showed that the native crystals had a packing defect, but that the defect was not present in crystals grown in the presence of methyl mercuric nitrate (a potential heavy-atom derivative – it would be five or six years before we could really begin to make use of heavy-atom derivatives in virus crystallography, so this was a somewhat romantic effort, but it led to the observation that allowed Jim Hogle to determine the TCV structure fifteen years later, using TBSV as a phase-determining starting point). After seeing the lysozyme model, I came to the naïve conclusion that the problems of protein structure had all been solved with myoglobin and lysozyme and that I should keep working on viruses. Aaron explained that he had agreed with Don Caspar that his laboratory would continue with TCV, while Don would pursue the closely related tomato bushy stunt virus (TBSV). Don had produced the first conclusive evidence for icosahedral symmetry of viruses from precession photographs of TBSV crystals, taken in 1956 at Birkbeck. As I planned to return to my graduate program at Harvard, I wrote to Don in the spring asking whether I could come to his laboratory to determine the structure of TBSV. He wrote back, saying that it was an exciting and important project; he probably should have written ‘far too challenging for a PhD thesis’, but fortunately he was at least as forward-looking and daring as I was starry-eyed and innocent.

My first serious experience with computer programming came when I needed to try to scale the (in retrospect, hopelessly inaccurate) intensities that I recovered with great difficulty from the very small-angle precession photographs I recorded from TCV crystals. Ken Holmes had written a program to scale the intensities from TMV layer-line diffraction, and he encouraged me to adapt it to single-crystal data. The critical matrix inversion involved an eigenvalue-filtering routine that Bob Diamond had implemented, so I learned some linear algebra to understand what I was doing. The subroutines I wrote came in handy later at Harvard, when I wanted to scale the much better data I could get from TBSV, using a Franks-mirror camera, as suggested by Don Caspar and as inspired, in part, by the Holmes-Huxley mirror-monochromator I had seen on the neighbouring X-ray generator.

Fast forward twelve years to February 1977. The laboratory had by then expanded toward Hills Road and the enlarged canteen, still the nerve center of the laboratory, was at the other end of the building. Computing was now ‘in house’, and the ‘computer girls’ no longer made their daily run to London. Michael Fuller’s stores continued to dispense almost anything you needed. Sydney’s worm project was well under way.

I had just completed collecting all the data needed to finish determining the TBSV structure at 2.9Å resolution. (Fortunately, I had not needed to get that far to complete a satisfactory PhD thesis.) David Blow had arranged a visiting fellowship in Trinity, providing not only more elegant accommodations than my student room in Peterhouse during the previous visit but the regular experience of Trinity High Table. A year before my return to Cambridge, Gerard Bricogne had visited us at Harvard to apply his direct-space averaging methods for non-crystallographic symmetry phase determination, and we had managed to achieve striking success with TBSV at 5.5Å. My task in the interval had been to acquire the data needed to see polypeptide chains and to help organise a computing workshop at the one location – the inter-regional computing center at Orsay – that Gerard knew could meet the requirements of the higher-resolution calculations. We planned to spend the spring preparing for that opportunity. I also decided to profit from the visit to get some hands-on instruction in electron microscopy from John Finch, trying to get good enough images of negatively stained P22 bacteriophage to calculate a 3-D reconstruction. (I did not succeed).

Gerard had built upon Tony Crowther’s analysis of non-crystallographic symmetry phase improvement, which had itself clarified many of the ideas implicit in the early efforts of Rossmann and Blow. One of Gerard’s key insights was that the reciprocal-space algorithms, which Tony had used to carry out trial calculations, would always be far too slow and that they had more readily computed, direct-space equivalents. He worked out an exceptionally elegant way of carrying them out on IBM OS360 computers, taking advantage of every byte of available core and every inch of available tapes and disks. His Fortran was as close to poetry as computer code can be. If most programs I used (and wrote) might be described as rambling, almost unreadable prose, Gerard’s code could be likened to a collection of haiku, annotated by precise and profoundly informative comments.

I had a desk back-to-back with John Finch, in a room with Joyce Baldwin and (I think) Tony Crowther. John and Aaron were busy working on the nucleosome structure. When Aaron realised that nucleosomes (often drawn in the years immediately following their discovery as schematic balls) had the shape of short cylinders, 55Å × 110Å, and that those dimensions could account for some of the well-known features of the small-angle diffraction pattern, he started talking about ‘platysomes’ – but fortunately, the term did not stick. The Finch et al paper published in September 1977 is a great classic, which I still use to teach the principles of X-ray diffraction, phase determination and electron microscopy.

Gerard worked at the maths labs to debug the programs for our summer workshop, and I joined him there on occasion to help put together some project-specific subroutines. When we were ready to transfer the computations to Orsay, we had a large cartful of punch cards and tapes which we drove to Dover, dragged onto the Calais ferry, and then transported to Paris (see Figure 10.1 – ferry transport was the 1977 version of FTP). The computations succeeded in producing a very fine map: to contour it, I had to return to Cambridge and use a program that made 35 mm films with the flying-spot scanner. A remarkably dedicated technical assistant printed the negatives on larger-format films, so that the contours were black on a transparent background, and I hurried back with them to Paris so we could tape them to plexiglass sheets, place dots, and trace the polypeptide chain. One surprise was the definition of the map: at 2.9Å resolution, one was not used to seeing bumps for many of the carbonyls. Ultimately, we realised that the iterative fivefold averaging (just three cycles, each of which took an hour of CPU and part of a week of preparation, plus a final round of averaging the map) had given us very accurate phases. It took over a year to build a Kendrew model in the Richards box we constructed for the purpose at Harvard, but I also knew the tedium of Allen screws and cathetometers would soon be obsolete, because Bob Diamond was working on a program called BILDER that would bring us to the frontier of computer graphics. Alwyn Jones was developing FRODO in Munich at the same time.

Figure 10.1. En route to the CECAM Workshop on Virus Crystallography. Gerard Bricogne and Stephen Harrison with programs, data and other luggage, boarding the ferry from Dover to Calais. Boxes of punch cards and 9-track tapes fill the floor of the wagon. July 1977. (copyright A. Olson, 2001)

Once part of the LMB community, subsequent short visits were rather like homecomings. Even after an interval of many years, Joy would remember that I took my tea without milk (perhaps a uniquely striking eccentricity, considering that the unbuffered canteen tea probably stained your oesophagus black). About a decade later, I happened to visit LMB to give a talk on our studies of repressor-operator complexes. Aaron was the host, and his very succinct introduction went something like ‘Steve was here in the laboratory twice – in 1965 and again in 1977. He will tell us about his recent work on the 434 phage repressor.’ A previous association with the laboratory was apparently the only biographical datum relevant to the occasion – and indeed, in many ways, it was.

REFERENCES

Finch et al, 1977. Structure of nucleosome core particles of chromatin. Nature 269, 29–36, 1977.

Olson, Bricogne and Harrison, Structure of TBSV. The Virus particle at 2.9 Å.

CHAPTER 11

From Virus Assembly to Signalling Between Bacterial Cells (1964–6)

Dale Kaiser

Department of Biochemistry, School of Medicine, Stanford University, Stanford, CA

Max Perutz’s LMB is the best example known to me of a large laboratory filled with many independent workers exchanging scientific ideas in the early years of molecular biology. The LMB has shown the way for theoreticians and experimental scientists with different skills and interests to collaborate productively. Perutz championed the free flow of ideas and felt that hierarchical relationships tend to impede that flow. Knowing that scientists like to talk over tea in small groups, he set up the fourth-floor canteen, run by his wife, Gisela, so as to eliminate distinctions of class. Everyone waited for their tea in the same cafeteria line. American postdocs, like me, benefited from Perutz’s scheme for it meant that we didn’t need to make an appointment to chat with someone outside the group in which we worked; we simply sat, uninvited, at their table for tea and talk. Although I spoke the language of molecular genetics, I was welcomed by biological physicists working on the structure of various proteins, because of the relation between DNA, as the genetic material, and the structure of proteins. It was a time when plausible, structural hypotheses for the regulation of gene expression were being made and tested.

In 1964, I brought my wife and two primary school-age children to Cambridge for an NSF-sponsored senior postdoctoral fellowship with Hugh Huxley and Aaron Klug. Steve Harrison was there at the same time working on the structure of turnip crinkle virus by X-ray crystallography. Perutz welcomed me to Peterhouse, his college, for several lunches. Hugh invited my wife and me to his rooms at King’s College to meet his friends and colleagues. Another attraction was the city of Cambridge with its shops and bookstores filled with geometry books related to structure. My aim was to learn electron microscopy, negative-staining, and how spherical viruses might be able to assemble themselves. The visit gave me the opportunity to learn the application of Caspar and Klug’s theory of quasi-equivalence for the symmetry of moderately large, spherical DNA viruses, like bacteriophage λ with icosahedral symmetry but more than 60 subunits. Figure 11.1 shows several paper models from the LMB archive of spherical structures having more than 60 subunits.

Figure 11.1. Collection of geometric models exploring possible virus structures etc. Aaron Klug and John Finch.

John Finch, a patient instructor, who had studied the structure of viruses by X-ray diffraction with Rosalind Franklin, taught me how to maintain and focus Huxley’s Siemens Elmiscop I. Returning from Cambridge in 1965 to Stanford University, I was able to set up a new EM facility for Arthur Kornberg’s biochemistry laboratory. EM was an important part of Kornberg’s demonstration of biologically-active DNA synthesis. I was also able to begin, with my own students and postdocs, to study how bacteriophage λ assembled new virus particles. Mutants of λ with a genetic defect in head assembly were able to make tails but no heads, and lysates of UV-induced lysogenic bacteria often contained intermediate structures. Even though λ head assembly might require a dozen proteins, its molecular basis could be approached because Jean Weigle had discovered that λ heads, produced by mutants defective in tail assembly, could spontaneously join to tails in vitro, to form complete and infective λ particles (Weigle, 1966). My efforts began with Sherwood Casjens, a graduate student, and Thomas Hohn, a postdoctoral fellow. They identified protein E as the major component of the head that accounted for 60 per cent of the mass of the head (Casjens et al, 1970), by joining radioactive heads to nonradioactive tails. Protein D was shown to be the second most abundant component, and it was missing from the small, empty protein spheres known as ‘petit λ’ (Casjens et al, 1970). This suggested an assembly pathway in which protein E formed a hollow shell that expanded with addition of protein D. Using in vitro assembly in cell extracts, proteins F and W were shown to add to DNA-filled head particles containing E plus D (as well as other head proteins), rendering them capable of attaching λ tails (Casjens et al, 1972).

The success of Casjens et al, 1972 with head protein assembly encouraged Terrie Masuda and me to attempt to package λ DNA in cell extracts. Using concentrated soluble extracts of E. coli cells (Wickner et al, 1972) that were lysogenic for λ (they carried λ as a provirus), we added pure, genetically marked λ DNA and observed the formation of morphologically complete and infective phage particles (Kaiser and Masuda, 1973). The phage assembled in vitro were sensitive to anti-λ antibody; they were DNAase-resistant, although DNAase added to the extract before λ DNA prevented their assembly. The phage particles that had been assembled were chimeras; they had the proper buoyant density and had a host-range characteristic of the genotype of the lysogen extracted, but the genotype of the particles formed in vitro was that of the DNA that had been added to the extract (Kaiser and Masuda, 1973). This experiment showed that the cutting and the packaging of DNA inside a head is coupled to the assembly of certain head protein subunits. In vitro packaging of λ DNA was significant, not only because it was the first DNA to be packaged in vitro, but also because it allowed each step in the packaging process to be investigated.

In 1971, I received a postcard from Max Delbruck, my PhD mentor, that said ‘Dear Dale, you have been working on λ long enough. Love, Max.’ Although we had made progress, I realised that for me the challenge of investigating λ assembly lay in working out the structural problems of virus morphogenesis. With that realisation and the graduation of most of the λ team, I decided to seek a multicellular bacterial system whose self-assembly could be studied in pure culture, trying to advance from multi-protein to multi-cellular assembly. In 1974–5, I took a sabbatical leave to work with Martin Dworkin in the department of microbiology at the University of Minnesota. The aim was to learn how to grow myxobacteria, to acquaint myself with their physiology, and try to introduce genetics for mutant studies. Dworkin is a first-rate bacterial physiologist, and we debated the merits of ‘holism’ and ‘reductionism’ over every lunch. Often the debate was joined by one of Dworkin’s professional philosopher colleagues. The challenge was to explain how each of the 45 different species of myxobacteria, which differ strikingly from each other, nevertheless self-assemble their own fruiting bodies (Reichenbach, 1984). For more than 200 years, microbiologists had distinguished the many species of myxobacteria by the forms of their fruiting bodies. Myxobacterial species distinctions survived the shift from purely morphologic classification to 16S RNA phylogenies (Sproer et al, 1999). Since the moderately complex shapes of fruiting bodies approach the more complex shapes of the internal organs of animals, the myxobacteria seemed to offer material that was easy to manipulate for experiments investigating how genes regulated organ assembly.

In the history of life on Earth, programs of multicellular morphogenesis appear to have evolved independently several times among the animals (King, 2004) and among plants and micro-organisms as well. Each single-celled organism that evolved a multicellular organism, like the choanoflagellate that is thought to have become a sponge, or the soil amoeba that became a slime mould, will have used for its multicellular development the set of cell-to-cell signals available to the single-celled organism (Kaiser, 2001). To see how multicellular morphogenesis may have evolved in myxobacteria, we set out to find which extracellular signals M. xanthus employs in developing its swarms and its simple, spherical spore-filled fruiting bodies. Two signals were identified from a set of conditionally defective mutants unable to form fruiting bodies by themselves (Hagen et al, 1978; LaRossa et al, 1983). However, when mutants were mixed with wild-type cells or with different mutants, some mixtures were able to form fruiting bodies with viable spores. Pairwise mixing of 57 mutants that were unable to develop led to identification of extracellular signals A and C. We found that medium conditioned by Myxococcus development contained a heat-stable and a heat-labile form of A-signal activity. Lynda Plamann, a postdoctoral fellow, found that heat-labile A-signal was a mixture of proteases and proteins that were degraded by those proteases (Plamann et al, 1992). Then Adam Kuspa, a graduate student, showed that heat-stable A-signal was a set of amino acids and small peptides containing those amino acids (Kuspa et al, 1992). It appears that certain amino acids are the primary A-signal molecules, while the extracellular release of proteases and proteins generates first peptides, then the specific A-signal amino acids.

When he was a postdoctoral fellow, Mitchell Singer found that fruiting body development can be induced by starvation and that A-signal helps M. xanthus assess the amount of nutrient available to support developmental protein synthesis (Singer and Kaiser, 1995). When Myxococcus is challenged by starvation, it must choose between initiating fruiting body development with differentiation of spores, or slow growth at a rate compatible with the level of nutrient still available. In nature, myxobacteria feed on particulate organic matter in the soil. Because either choice will kill the vast majority of cells (more than 99 per cent), the better option depends on a cell’s projected estimate of future nutrient availability. If local nutrient is on its way to exhaustion due to consumption by competing soil microbes, then slowing growth to match the level of residual nutrient leads to slower and slower growth, until Myxococcus starves to death. Colin Manoil found that the limited availability of any amino acid, or starvation for carbon, energy or phosphorus induces fruiting body development (Manoil and Kaiser, 1980), while Kimsey found that neither purine nor pyrimidine starvation could induce development (Kimsey and Kaiser, 1991). Since a complete set of amino-acylated tRNAs is needed for protein synthesis, the absence of one or more amino-acylated tRNA is readily perceived. In M. xanthus, as in many other bacteria, the absence or shortage of any one of the charged tRNAs being called for by a messenger RNA causes a ribosome to synthesise guanosine tetra- (and penta-) phosphate, (p)ppGpp. Pyrophosphate, or P~P, is transferred from ATP to GTP, and a stringent response is triggered. Singer showed that (p)ppGpp was both necessary and sufficient to initiate fruiting body development (Singer and Kaiser, 1995). Giglio et al, 2011 found that development is initiated then propagated by a sequence of enhancer binding proteins, acting at sigma54 promoters that respond to the level of A-signal. Recently, Sarwar confirmed that M. xanthus uses a stringent response and the cell-density-dependent A-signal to predict the future nutrient availability, a sophisticated task (Sarwar and Garza, 2012).

C-signal tells a different story. C-signal is cell-bound, and signal exchange requires cell-cell contact, unlike the diffusible A-signal. Active C-signal was purified from the membrane fraction of whole cells, using a detergent and a bioassay of restoring aggregation and sporulation, in vitro, to a mutant lacking the csgA gene (Kim and Kaiser, 1990c). Mutants, lacking csgA, arrested their development without aggregating (Kaiser, 2003), and the csgA mutants formed very few, if any, spores. The sporulation background was low enough to construct a sensitive rescue assay for C-signal. A 17 kDa protein was purified from starved wild-type cells that, by addition, restored the capacity of csgA mutants to develop and to sporulate (Kim and Kaiser, 1990c). C-signal was not recovered from cells that hadn’t been starved, or from extracts of csgA mutants (Kim and Kaiser, 1990a). Subsequent experiments confirmed that the 17 kDa protein is the signal, and that it is made by the action of a cell-surface protease on an outer-membrane protein. C-signal carries information as to cell density and to cell orientation with respect to other cells. Surprisingly, it was found that non-motile mutants of M. xanthus arrested their fruiting body development at the same stage as did the csgA mutants. This surprising result suggested that C-signalling might occur only between aligned cells brought into end-to-end contact by cell movement (Kroos et al, 1988). Seung Kim tested this hypothesis by mechanically placing non-motile cells into end-to-end alignment (Kim and Kaiser, 1990b). The asymmetry of the long rod-shaped M. xanthus cells was used to orient them lengthwise as they tumbled from suspension into the narrow grooves produced by scoring an agar gel with fine-grained, aluminum oxide abrasive paper. Phase contrast microscopy revealed that cells, which had settled into the grooves, were oriented with their long axes parallel to the axis of the groove and that they were in end-to-end contact (Kim and Kaiser, 1990b). However, because the grooves were 5–10 µm wide, cells were also found lying side-by-side in the grooves, and the experiment did not exclude C-signalling between the sides of cells. Experiments with narrower grooves will be required to test whether side-by-side contacts are involved in C-signalling, as end-to-end contacts seem to be. Meanwhile, Thomas Gronewold, a postdoctoral fellow, discovered positive feedback in the C-signalling circuit that is controlled by the act operon of five co-transcribed genes (Gronewold and Kaiser, 2001; Gronewold and Kaiser, 2002). That feedback progressively raises the number of C-signal molecules per cell from a few at three hours post-starvation to several hundred by eighteen hours (Kim and Kaiser, 1990a; Gronewold and Kaiser, 2001). Apparently, the rise serves to coordinate C-signal-dependent gene expression; each developmental gene has its own particular threshold for expression. C-signal also triggers simultaneous sporulation and loss of cell motility when cells no longer need to move (Kroos and Kaiser, 1987; Julien et al, 2000). C-signal is a morphogen.

As M. xanthus grows on a solid nutrient medium like agar, it elaborates a swarm whose cells are constantly moving. However, the movements are well organised and the cells never seem to jam up even though collisions are frequent. Apparently, they move constantly after they have grown to a high cell density so that all cells have access to oxygen from the atmosphere above and to carbon, nitrogen, and phosphate nutrient from the agar medium below. When M. xanthus has enough nutrient to grow instead of developing fruiting bodies, it shuffles about in the swarm. The long, rod-shaped cells power movement only in the direction of their long axis. Cells move this way for hydrodynamic reasons and because their motility engines are located at both cell poles. There are type IV pili at the front to pull cells forward, and slime secretion engines at the back to push cells forward. Forward movement is punctuated with reversal and equal movement in the opposite direction. An oscillator, known as the ‘pacemaker’, causes each cell to reverse direction every 8–9 min (Kaiser and Warrick, 2011). The periodic reversals are essential for M. xanthus to swarm; when reversals are blocked, a swarm cannot expand (Wu et al, 2009). The organisation of a swarm is such that each cell appears to move at random without attenuating the movement of its neighbours. This consistent behaviour might be considered the first law of swarming. The product is a circular swarm that expands at a constant rate, unlike a colony that expands at an ever-decreasing rate as nutrient and oxygen are consumed. Even when swarming at rather high density, an 0.5 mm-wide ring of cells at the swarm edge are growing as fast as cells can grow in aerated liquid culture. The organised behaviour of an M. xanthus swarm is one that supports rapid growth.

Evidence for a timing signal is found in the behaviour of two sorts of highly organised, multicellular structures at the edge of an expanding swarm: the rafts and the mounds. Rafts are dense, single-layered, rectangular assemblies of hundreds of cells that have their long axes in parallel with each other. Although they are packed together, raft cells slide smoothly alongside each other. Mounds are round, multi-layered assemblies of thousands of cells. The layers are nested one on top of another. Mound cells have two degrees of freedom. They can move rapidly from one layer to the next, either up or down. Alternatively, cells can move more slowly to a new position within the same layer. It is believed that mounds are built from the bottom up, and for that reason cells in the top layer will have been mound residents longer than any other cells (Kaiser and Warrick, 2013).

Every cell has its own pacemaker, an oscillator that causes the cell to reverse its gliding direction regularly, every 8–9 minutes (Kaiser and Warrick, 2011). The new-found signal is proposed to bring the pacemakers in a pair of cells, which are signalling to each other, to the same phase of their reversal cycle, and thus synchronised with each other. Moreover, once synchronised, two pacemakers remain synchronised. The signal was discovered when viewing time-lapse movies of cell movement at the swarm edge. After a one-hour delay, all cells in the top (fifth) layer of a mound ‘exploded’. All of a sudden they increased their speed of movement more than threefold to 12 µm/min, and within a minute decreased their speed threefold back to the 4 µm/min speed average. The timing signal is thought to spread by transient aligned contact between pairs of adjacent cells. Accordingly, the hour’s delay is the time required to spread the signal among all cells in the mound’s top layer.

The signal is thought to be transferred from one cell to another by multi-protein structures called ‘focal adhesions’ (Mignot et al, 2005; Mignot et al, 2007). Focal adhesions are found as a series of discrete foci running along one side of a cell from its leading end to just forward of mid-cell. Focal adhesions were first proposed as complexes of motor proteins (Nan et al, 2011; Sun et al, 2011). But it has been pointed out that the fifteen or more proteins that characterise an adhesion are more suited for signalling than for motorising cells (Kaiser and Warrick, 2013). One reason for proposing them as signalling proteins is their apparent capacity to bind to each other in pairs (Nan et al, 2010). Another reason is their apparent localisation in different membrane-bound compartments of M. xanthus cells. Proceeding inward, the compartments are defined by the outer membrane, the periplasm, the outer surface of the peptidoglycan sacculus, the inner membrane, and finally the cytoplasm of these Gram-negative bacteria. Accordingly, the signal could link oscillating methylated-FrzCD in the pacemaker of one cell mechanically through pairs of protein 1 transiently bound to protein 2 links to CglB – one of the fifteen motility proteins found in an adhesion – on the surface of that cell. (CglB was discovered by Jonathan Hodgkin, formerly on the LMB staff, in 1979 when he was a postdoctoral fellow at Stanford. He also discovered that it seemed to have a role in signalling.) It is proposed that CglB on the surface of the first cell would assemble with CglB on the surface of the second cell. From CglB the signal would link through the same series of protein 1• protein 2 pairs until it reached FrzCD in the pacemaker of the second cell. When completed, the series of signal links would bring the pacemakers of both cells to the same, average phase of their oscillatory cycles. The signal is still a work in progress. At least, focal adhesions seem to create a new force that causes pairs of cells to adhere to each other, transiently.

REFERENCES

Casjens, S., T. Hohn and A.D. Kaiser, 1970. Morphological proteins of phage lambda: identification of the major head protein as the product of gene E. Virology 42, 496–507.

Casjens, S., T. Hohn and A.D. Kaiser, 1972. Head assembly steps controlled by genes F and W in bacteriophage lambda. Journal of Molecular Biology 64, 551–63.

Gronewold, T.M.A. and D. Kaiser, 2001. The act operon controls the level and time of C-signal production for M. xanthus development. Molecular Microbiology 40, 744–756.

Gronewold, T.M.A. and D. Kaiser, 2002. act operon control of developmental gene expression in Myxococcus xanthus. Journal of Bacteriology 184, 1172–9.

Hagen, D.C., A.P. Bretscher and D. Kaiser, 1978. Synergism between morphogenetic mutants of Myxococcus xanthus. Developmental Biology 64, 284–96.

Julien, B., A.D. Kaiser and A. Garza, 2000. Spatial control of cell differentiation in Myxococcus xanthus. Proceedings of the National Academy of Science of the United States of America 97, 9098–9103.

Kaiser, D., 2001. Building a multicellular organism. Annual Review of Genetics 35, 103–23.

Kaiser, D., 2003. Coupling cell movement to multicellular development in myxobacteria. Nature Reviews Microbiology 1, 45–54.

Kaiser, D. and T. Masuda, 1973. In vitro assembly of bacteriophage lambda heads. Proceedings of the National Academy of Science of the United States of America 70, 260–64.

Kaiser, D. and H. Warrick, 2011. M. xanthus swarms are driven by growth and regulated by a pacemaker. Journal of Bacteriology 193, 5898–5904.

Kaiser, D. and H. Warrick, 2013. Focal adhesions could be cell-cell signaling complexes in myxobacterial swarms. Manuscript in preparation.

Kim, S.K. and D. Kaiser, 1990a. C-factor: A cell-cell signalling protein required for fruiting body morphogenesis of M. xanthus. Cell 61, 19–26.

Kim, S.K. and D. Kaiser, 1990b. Cell alignment required in differentiation of Myxococcus xanthus. Science 249, 926–8.

Kim, S.K. and D. Kaiser, 1990c. Purification and properties of Myxococcus xanthus C-factor, an intercellular signaling protein. Proceedings of the National Academy of Science of the United States of America 87, 3635–9.

Kimsey, H.H. and D. Kaiser, 1991. Targeted disruption of the Myxococcus xanthus orotidine 5′-monophosphate decarboxylase gene: Effects on growth and fruiting-body development. Journal of Bacteriology 173, 6790–97.

King, N., 2004. The unicellular ancestry of animal development. Developmental Cell 7, 313–25.

Kroos, L., P. Hartzell, K. Stephens and D. Kaiser, 1988. A link between cell movement and gene expression argues that motility is required for cell-cell signalling during fruiting body development. Genes and Development 2, 1677–85.

Kroos, L. and D. Kaiser, 1987. Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions. Genes and Development 1, 840–54.

Kuspa, A., L. Plamann and D. Kaiser, 1992. Identification of heat-stable A-factor from Myxococcus xanthus. Journal of Bacteriology 174, 3319–26.

LaRossa, R., J. Kuner, D. Hagen, C. Manoil and D. Kaiser, 1983. Developmental cell interactions in Myxococcus: analysis of mutants. Journal of Bacteriology 153, 1394–1404.

Manoil, C. and D. Kaiser, 1980. Accumulation of guanosine tetraphosphate and guanosine pentaphosphate in Myxococcus xanthus during starvation and myxospore formation. Journal of Bacteriology 141, 297–304.

Mignot, T., J.P. Merlie and D. Zusman, 2005. Regulated pole-to-pole oscillations of a bacterial gliding motility protein. Science 310, 855–7.

Mignot, T., J. Shaevitz, P. Hartzell and D. Zusman, 2007. Evidence that focal adhesions power bacterial gliding motility. Science 315, 853–6.

Nan, B., J. Chen, J. Neu, R. Berry, G. Oster and D. Zusman, 2011. Myxobacteria gliding motility requires cytoskeleton rotation powered by proton motive force. Proceedings of the National Academy of Science of the United States of America 108, 2498–2503.

Nan, B., E. Mauriello, I.-H. Sun, A. Wong and D. Zusman, 2010. A multi-protein complex from Myxococcus xanthus required for bacterial gliding motility. Molecular Microbiology 76, 1539–54.

Plamann, L., A. Kuspa and D. Kaiser, 1992. Proteins that rescue A-signal-defective mutants of Myxococcus xanthus. Journal of Bacteriology 174, 3311–18.

Reichenbach, H., 1984. Myxobacteria: a most peculiar group of social prokaryotes. In Myxobacteria (ed. E. Rosenberg), pp. 1–50, Springer-Verlag, New York.

Sarwar, Z. and A.G. Garza, 2012. The Nla28S/Nla28 two-component signal transduction system regulates sporulation in Myxococcus xanthus. Journal of Bacteriology 194, 4698–4708.

Singer, M. and D. Kaiser, 1995. Ectopic production of guanosine penta- and tetraphosphate can initiate early developmental gene expression in Myxococcus xanthus. Genes and Development 9, 1633–44.

Sproer, C., H. Reichenbach and E. Stackebrandt, 1999. Correlation between morphological and phylogenetic classification of myxobacteria. International Journal of Systematic and Evolutional Biology 49, 1255–62.

Sun, M., M. Wartel, E. Cascales, J. Shaevitz and T. Mignot, 2011. Motor-driven intracellular transport powers bacterial gliding motility. Proceedings of the National Academy of Science of the United States of America 108, 7559–64.

Weigle, J., 1966. Assembly of phage lambda in vitro. Proceedings of the National Academy of Science of the United States of America 55, 1462–6.

Wickner, W., D. Brutlag, R. Schekman and A. Kornberg, 1972. RNA synthesis initiates in vitro conversion of M13 DNA to its replicative form. Proceedings of the National Academy of Science of the United States of America 69, 965–9.

Wu, Y., D. Kaiser, Y. Jiang and M. Alber, 2009. Periodic reversal of direction allows myxobacteria to swarm. Proceedings of the National Academy of Science of the United States of America 106, 1222–7.

CHAPTER 12

Me and Hills Road (1964–6)

Frank Stahl

Institute of Molecular Biology, University of Oregon, Eugene, OR

Figure 12.1. Frank and Henriette (Jette) Foss.

Figure 12.2. Frank and Matt Meselson.

WHY CAMBRIDGE?

In late summer 1964, in search of an introduction to proteins in the learned company of J.D. Smith, Sydney Brenner and Francis Crick, I embarked from New York en famille (on the SS France) for Southampton. While my PhD background in formal phage genetics and my postdoctoral collaboration with Matt Meselson convinced me that understanding of life at the molecular level was possible, it was evident that my familiarity with proteins (or chemistry of any kind) was not what it might have been.

A BIG MISTAKE

Within a week after arrival, I realised that my aim was way off. I couldn’t get my mind around a balky fraction collector, and the cold room did not attract. I quit. Sydney said that was typical of visiting American geneticists seeking biochemical savvy. (On a later occasion Sydney again described American postdocs: ‘They come to Cambridge loaded with powerful new techniques but in search of ideas, which they expect to get from me.’ When I protested, ‘Hey, I’m an American’, he responded (more or less): ‘Well, you certainly are not.’)

HANGING OUT

To fill the days that followed my defection, I discussed genetics with Roger Freedman and Jan Drake, played soccer with Glauco Tocchini-Valentini, bicycled on the wrong side of the street from Conduit Head to Hills Road, marvelled at John and Lesley Menninger’s thatch, walked the Roman Road, punted on the Cam, drank Irish coffee to combat the drizzle, slept through classy performances at the Aldwych and supported Johnson against Goldwater among overseas Americans.

SERENDIPITY I

One day, during an early autumn stroll, Dick Epstein told me some stories about the newly discovered ‘amber’ mutants of a virus that infects bacteria (Epstein et al, 2012). Because it is simple and multiplies rapidly, this virus, called T4 phage, is a terrific object for investigating the most basic life processes. The amber mutants have the remarkable property that they can occur in every gene of the virus. When they occur in an ‘essential’ gene, they block viral growth in their ordinary host, E. coli strain B. Importantly, the mutants grew well in another E. coli strain (K). (That the mutants occurred in many different genes was demonstrated by the simple observation that most pairs of these mutants could complement, i.e. cooperate with, each other to promote growth in strain B.) The amber mutants were hugely valuable for analysing viral development. For each one of the mutants, Dick and his collaborators asked, ‘Why can’t the amber mutant grow when it infects strain B? What is it that the amber mutant cannot do that the ordinary T4 can do?’ The answers to these straightforward questions identified a number of discrete gene functions required for growth. The amber mutants, in conjunction with Bob Edgar’s temperature-sensitive mutants, revealed that T4 genes are conspicuously clustered by function (Epstein et al, 1964). In E. coli, the bacterial host of T4, genes are similarly clustered according to function. The bacterial gene clusters, called operons, serve to coordinate the expression of functionally related genes by providing for their transcription into a single RNA messenger. It is a property of this mode of coordinated control that amber mutants in the ‘upstream’ gene in such an operon have ‘polar’ effects – they not only eliminate the function of that gene but, according to the French school, also reduce the function of the ‘downstream’ genes in the same operon. During our perambulation, Dick confessed that none of his T4 amber mutants seemed to act like ‘polar mutations’. A polar mutation in T4 would have been recognised by its failure to complement mutants, either temperature-sensitive or amber, in any of the genes in the cluster. Their apparent absence cast doubt on the otherwise attractive idea that the T4 genes were clustered to facilitate their coordinated regulation.

IDEA

If the clustering of functionally related genes of T4 does not reflect co-regulation, perhaps it reflects ‘co-adaptation’. In discussing this thought with Leslie Barnett (a font of knowledge and pillar of sensibility in the Crick/Brenner unit of the LMB), I learned that R.A. Fisher had earlier proposed that, in populations with appreciable heterozygosity, co-adapted genes would be subject to separation by ordinary, homology dependent recombination. The loss of vitality resulting from this separation would select for reduced recombination between them, favoring tight linkage. According to Leslie, geneticists never found much evidence supporting Fisher’s proposal. Of course, in the diploid eukaryotes studied in those days the selective force Fisher identified might have been too weak to play a conspicuous role in determining gene order. In T-even phages, however, the selective forces might be decisively stronger. I liked the idea because it was counter-cultural and because personal experience had taught me that the plaques made by the progeny of a T4 × T2 cross comprised a zoo, with the extremely small size of some plaques suggesting disruption of seriously co-adapted genes.

TESTING THE HYPOTHESIS

A test of the idea was immediately obvious. We needed (1) a set of Dick’s amber mutants, one for each of the T4 genes so far identified and (2) a bunch of amber mutants in T2. Support for the hypothesis would require finding two complementing ambers in T4 and two complementing ambers in T2 that failed to complement in all four inter-type combinations. The demonstration would be complete when the two genes of such mutant pairs were seen to be in the same gene cluster. A positive outcome of the project would provide a clear illustration of an important evolutionary concept regarding the moulding of a genome. Better yet, it could be performed by elementary phage methodology: spot tests on seeded top layers of Petri plates. No fraction collector, no cold room. Only one thing was missing: a pair of hands to keep my hands company during what promised to be a straightforward but repetitive procedure.

SERENDIPITY II

Then, late one afternoon, Noreen Murray, an acquaintance with a background in fungal genetics, appeared in my lab and asked if there was anything going on that she might help with. She wanted to take part time off from her work in the botany department if I had anything for her. Did I! The search for co-adapted genes would use (over and over) all the elementary moves of phage genetic analysis, making it an ideal project for a beginner, especially for one familiar with sterile technique. Noreen signed on once I convinced her that her lack of experience would be more of an asset than a problem.

SERENDIPITY III

At this point, Sydney volunteered that, in the cold room, there was a mutagenised stock of T2 from which we could isolate amber mutants. The hunt was on, and, with the expedition characteristic of phage genetics, we soon identified a pair of genes for which complementation of the two T4 mutants was perfect and of the two T2 mutants was likewise perfect, but between T4 and T2 was stunningly negative in both directions. Epstein’s T4 map showed that these genes were immediately adjacent on the linkage map, in the tail fibre region. We had our illustration and we wrote a rarely cited classic (Stahl and Murray 1966).

CONTINUING BENEFITS

During the spot-testing for complementation we noted some T4 gene pairs for which complementation looked feeble. In some cases, this result was supported by more quantitative, one-step growth experiments (Ellis and Delbrück 1939). Could the poor complementation of these mutant pairs, in fact, signal polarity? Subsequent tests, conducted following my return to Eugene, confirmed that possibility and led to the development of simple, one-step growth tests for determining the direction of transcription of a number of pairs of adjacent genes (Stahl et al, 1966). (It wasn’t the last time that I tried to distinguish between two hypotheses and ended up receiving satisfying support for both.) The work kept me occupied for several years (Nakata and Stahl 1967; Stahl et al, 1970), following which I left T4 for phage Lambda, whose lifestyle allowed one to track simultaneously the transfer of matter and information from parents to individual progeny particles, using various adaptations of a protocol developed by Matt Meselson and Jean Weigle (1961).

SUMMING UP

My sojourn in the Laboratory of Molecular Biology acquainted me with the deservedly famous ambience and personnel of that institution. I learned that protein chemistry didn’t need me, that King’s College High Table was for tougher folk than I, and that Sydney talked but Francis conversed. The entire, memorable experience was topped up by the opportunity to work with Noreen years before she was honoured for her contributions to phage-based genetic engineering. Unforgettably, on my sabbatical leave at LMB I had the pleasure of teaching Noreen Murray (1935–2011) the ABC of phage genetics using Dick Epstein’s (1929–2011) amazing amber mutants. How blessedly lucky can a guy be? Serendipity indeed!

REFERENCES

Edgar, R.S. and I. Lielausis, 1964. Temperature-sensitive mutants of bacteriophage T4D: their isolation and genetic characterization. Genetics 49, 649–62.

Ellis, E.L. and M. Delbrück, 1939. The growth of bacteriophage. Journal of General Physiology 22, 365–83.

Epstein, R.H., A. Bolle, C.M. Steinberg, E. Kellenberger, E. Boy de la Tour, R. Chevalley, R.S. Edgar, M. Susman, G.H. Denhardt and A. Lielausis, 1964. Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symposia on Quantitative Biology 28, 375–94.

Epstein, R.H., A. Bolle and C.M. Steinberg, 2012. Amber mutants of bacteriophage T4D: their isolation and genetic characterization. Genetics 190, 831–40.

Meselson, M. and J.J. Weigle, 1961. Chromosome breakage accompanying genetic recombination in bacteriophage. Proceedings of the National Academy of Science of the United States of America 47, 857–68.

Nakata, A. and F.W. Stahl, 1967. Further evidence for polarity mutations in bacteriophage T4. Genetics 55, 585–90.

Stahl, F.W. and N.E. Murray, 1966. The evolution of gene clusters and genetic circularity in microorganism. Genetics 53, 569–76.

Stahl, F.W., N.E. Murray, A. Nakata and J.M. Crasemann, 1966. Intergenic cis-trans position effects in bacteriophage T4. Genetics 54, 223–32.

Stahl, F.W., J.M. Crasemann, C. Yegian, M.M. Stahl and A. Nakata, 1970. Co-transcribed cistrons in bacteriophage T4. Genetics 64, 157–70.

CHAPTER 13

‘What Are You Working On?’ A Californian in Cambridge (1965–6)

Harry F. Noller

Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California at Santa Cruz, CA

During my final year of graduate school in the Institute of Molecular Biology at the University of Oregon, I received notification that I had been awarded a three-year NIH postdoctoral fellowship to work at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. That same week, I received another tempting offer: to go on the road with the Benny Wilson Blues Band. On the road with Benny, I reasoned, there would be no science, but I was fairly confident that there would be music in Cambridge. Accordingly, I accepted the NIH fellowship and gave Benny my regrets. My decision panned out – there was of course plenty of exciting science in Cambridge. And I found a jazz group.

When I arrived in Cambridge in November 1965, I took the bus down Hills Road to the MRC (as we then called it) to see the lab. The receptionist welcomed me with ‘Oh, Dr Noller! Let me see if I can find Dr Perutz!’ I was certain that she must have made a colossal mistake – she had taken me for some other, distinguished Dr Noller. I tried to come to grips with the idea that we were going to search for Max Perutz, the founder of protein crystallography, director of the MRC Laboratory for Molecular Biology and winner of the 1962 Nobel Prize in Chemistry. He was not in his office or in his lab. As we walked along the hall on the first floor, the cold room door swung open, and out stepped a small bald man wearing a white coat and National Health Service glasses, carrying an Erlenmeyer flask containing a red liquid. ‘Dr Perutz, this is Dr Noller’, said the receptionist. I took a deep breath. Max stopped and smiled shyly, extending his hand, welcoming me to Cambridge and to the MRC. Time seemed to be of no consequence as we chatted. Condensation slowly formed on the surface of the cold Erlenmeyer. I was stunned: Max Perutz was doing a haemoglobin prep. Himself. And he had stopped in the middle of his prep to chat with me. I could not shake the feeling that something was terribly wrong, that I was a fraud: Max could not possibly know who I was, or care.

In the following weeks, intimidation by the shadows of the giants of Cambridge science, from Newton to Crick, continued to affect me. Struggling to blend into Cambridge life, I began to wear a tweed jacket and tried to disguise my accent and even my loping California walk. Deprived of my daily morning shower I took refuge in the bathtub, where I was monitored closely by our landlady, Miss Hicks, vigilant against the splashing of water into the overflow and thence into her garden. ‘Mind the baaath!’ echoed the warnings from below, as I tightened my grip on Ken Kesey’s Sometimes a Great Notion for confirmation of the continued existence of America. I even pretended to understand the bus conductor when he asked for my fare in a dialect from an unknown crevice somewhere deep in the English language tree. In my confusion, Paul Sigler helped me open my eyes to my native culture. Paul was a graduate student in his thirties at the MRC, working with the crystallographers David Blow and Brian Matthews on the structure of chymotrypsin in a dimly lit, cramped corner of the basement. He had played football at Princeton and had left medicine to become a scientist after completing his MD and residency at Columbia. One afternoon, Paul was charging down the hallway in front of me, the collar of his unbuttoned, billowing white lab coat turned up in the style of a 1950s urban delinquent, chewing his gum aggressively, his stride long and confident. His presence dominated the hallway. As he passed Max Perutz, he smiled and gave Max a powerful slap on the back, with a loud ‘Howya doin’, Max?’ Smiling politely, Max absorbed the blow and mouthed a greeting to Paul, recovering his wind. It was possible to be simultaneously American and in Cambridge.

Adjusting to life in the exotic outside world of England was no less intimidating. To this end, we bought a pretty little green 1947 MG YB saloon, a sort of miniature Bentley, for £30. On the first nice weekend, we took it for a drive in the country. Cruising down a narrow lane bordered by picturesque hedgerows and country manors, we came around a curve to find ourselves face-to-face with a madman driving straight at us, on direct course for a head-on collision. As the car approached, I could make out the terror-stricken expressions on the faces of its occupants, which came with the realisation that I was on the wrong side of the road. Recovering from this near-death experience, we headed back to Cambridge to find a parking place near our apartment. I was cheered by our good fortune in finding a convenient spot near the corner of Lensfield Road and Hills Road. As I was locking the car, I noticed the signal at Hills Road turn green. All of the cars parked in front of us began to move forward, just as the cars behind us began honking their horns.

My postdoctoral advisor was the delightful Welshman Ieuan Harris, who had learned protein chemistry in Cambridge in the biochemistry department on Tennis Court Road, sequencing peptides and proteins by the methods that Fred Sanger had used to determine the amino acid sequence of insulin, for which he received his first Nobel Prize. As a graduate student, I had worked on the serine protease subtilisin, and my advisor Sidney Bernhard had suggested that I move up the ladder in complexity for my postdoctoral research by studying the more challenging redox enzyme glyceraldehyde-3-phosphate dehydrogenase (GPDH), the focus of Ieuan’s lab. One reason for sequencing GPDH was that it formed very high-quality crystals that diffracted to around 1.5Å; in fact, the enzyme was purified by ammonium sulphate crystallisation from a crude extract of lobster muscle (incidentally providing the starting materials for a delicious bisque). Heavy-atom derivatives had been prepared and crystallised by Herman Watson, and high-resolution X-ray data were being collected to solve the structure. Sequencing the protein – at 333 amino acids, the longest polypeptide chain ever attempted at the time – would provide crucial information for fitting the structure to the electron density map, so our work would put us right in the middle of the excitement of determining the first structure of an enzyme containing an NAD co-factor. Or so we imagined.

In Ieuan’s lab, we benefited tremendously from working next door to Brian Hartley. Brian and his group had developed powerful yet simple micro-scale methods for sequencing proteins, centered around their revolutionary Dansyl-Edman method. Small amounts of protein were digested with trypsin or chymotrypsin and the resulting peptides separated by two-dimensional paper electrophoresis and sequenced by the Dansyl-Edman technique. However, we found that some larger peptides failed to behave in the paper electrophoresis procedure, and so we resorted to using ion-exchange columns at my suggestion and to Ieuan’s consternation. I had the good fortune of being able to work together with the very talented Australian postdoc Barrie Davidson. Barrie shared an acute sense of the absurd with Ieuan’s red-headed Welsh graduate student Graham Jones; together, they were responsible for an almost constant stream of low-level laughter in the lab, while doing lots of experiments that actually worked. Our use of ion-exchange chromatography to separate the tryptic and chymotryptic peptides, à la Moore and Stein, was not received well by Ieuan, who tried to persuade us to use just a Sephadex sizing column followed by paper electrophoresis, a simpler approach that he himself had developed. The high concentrations of urea that we were using, in an attempt to recover some of the poorly soluble peptides, came under more serious fire when Ieuan discovered a cluster of urea crystals clogging the flow-cell of his precious UV detector, and spilling over onto the floor. While cleaning up the mess, with Ieuan safely out of earshot, Barrie and Graham burst into song, set to the melody of Bernstein’s ‘Maria’, from West Side Story:

‘U-re-a!

I just made eight molar u-re-a!

And, suddenly I found,

My H-bonds came unbound …’

Life outside the lab around the university was no less intriguing. My wife Betty became involved in the theatre life of Cambridge, appearing in a production of Jean Genet’s The Balcony. I joined an interesting group of musicians who played on Thursday nights at a jazz pub called The Lion. The leader, pianist Marty Gellen, was from LA, where he had played in a hit pop group called The Teddy Bears with the notorious Phil Spector. The bassist, Daryl Runswick, was a Cambridge undergraduate who went on to a career as musician and composer, working with artists as diverse as Sarah Vaughn, Pierre Boulez and Elton John. Tony Bicât, the drummer, another undergraduate, became a successful film director, writer and composer for British television. We even performed in the 1641st concert of the austere Cambridge University Musical Club (Figure 13.1), where half the audience rose as one and exited the hall when it became clear from the first two bars of John Coltrane’s ‘Cousin Mary’ that we would not be playing chamber music. But I did manage to play chamber music at our apartment with Bill Lipscomb, the Harvard boron hydride chemist, protein crystallographer and accomplished clarinettist, who was on sabbatical in Cambridge. At Christmastime, nearly the whole lab pulled together to rehearse Bach’s Magnificat in the evenings in the cafeteria on the roof of the MRC, which we performed at one of the colleges for a small, unlucky gathering of family and friends.

Figure 13.1. Programme from the 1641st concert of the Cambridge University Musical Club. The correct date was actually 5 February 1966.

Daily life at the MRC was structured around three events in which everyone was expected to participate: morning coffee, lunch and afternoon tea, which all took place in the cafeteria, built with help from funds donated by Max from his Nobel Prize money. This was where you could test and exchange ideas, trouble-shoot experiments and learn first-hand about the revolutionary science that was emerging from this remarkable place. At one table, you might find Francis Crick, Sydney Brenner, John Smith and Brian Clark; at the structure table, Max Perutz, John Kendrew, Hugh Huxley, Brian Matthews, David Blow, Richard Henderson and Aaron Klug; at the protein-chemistry table, there was Fred Sanger, Ieuan Harris, César Milstein and Brian Hartley. These gods were actually having lunch together, along with the handful of postdocs who could summon the courage to sit down with them. Among the postdocs was a large contingent of talented Americans, including John Abelson, Howard Goodman, Jim Dahlberg, Tom and Joan Steitz, Bob Kretsinger, Harvey Lodish, Art Landy, Bob Heinrikson and Paul Sigler (who was actually a graduate student), who would go on to have successful careers and become leaders of American science. Among the other impressive international students and postdocs were Glauco Tocchini-Valentini (from Italy), Anand Sarabhai (from India), Kjeld Marcker (from Denmark) and César Milstein (from Argentina). Even the technicians – like Bart Barrell, who helped Fred Sanger develop his RNA and DNA sequencing methods, and John Jarvis, who, when not playing rugby, worked with César Milstein to sequence the first antibody protein chains and develop monoclonal antibodies – had a major impact on the development of this new science.

The MRC fostered a strong ethic of frank, critical discussion – no secrets, and no polite pulling of punches if you didn’t believe someone’s experiment. There was a certain amount of arrogance, but an equal and opposing tradition of cutting people down to size. Cambridge scientists thought very big (Perutz: If you can solve the crystal structure of a small molecule, why not an enormous protein?) and they thought small (Sanger: If you can determine what the first amino acid in a chain is, can you determine the second?) in ways that became very big. Naïve questions asked over coffee led to paradigm-breaking discoveries. As a young postdoc, you could look around the cafeteria and see the giants of molecular biology, still in their prime, drinking tea and telling jokes. You could say, ‘This is it’, and it made doing exciting science seem suddenly a real possibility, because you could understand that these giants were also mere human beings, with food stains on their shirts and common mistakes in their scientific conversations.

One difference between the MRC and American science cultures of the time was the absence of regular weekly research seminars. Instead, there was an annual event when everyone at the MRC presented a short talk in the seminar room. One exception was during a visit by a prominent American scientist, who invited himself to give a seminar. As he began his talk, Sydney Brenner took a seat in the front row, but turned to face the audience. During the lecture, Sydney provided a spontaneous and scathingly witty commentary, effectively neutralising the American’s efforts to impress the Cambridge crowd. More typical were informal evening presentations, such as one given by Fred Sanger in a small meeting room in King’s College. With his trademark modest delivery, Fred showed the results of a new ‘fingerprinting’ method he had developed for sequencing RNA, based on a two-dimensional paper electrophoresis separation of RNase T1 oligonucleotides, in which RNA fragments of related sequence migrated in geometric ‘graticules’ depending on their U, A and C content. This was characteristic of the Cambridge style – revolutionary methods and ideas worked out in an apparent vacuum, betraying little sense of competition or influence from the outside world.

Sanger was head of the division of protein chemistry (although much of the research in his division was occupied with research on RNA and even DNA), but anyone wanting to talk to Fred had to stand next to him in his small separate laboratory and wait for a pause in his experiment. He worked in the lab every day from 9 to 5 for his entire career, even after two Nobel Prizes, virtually by himself (usually helped by only a single technician) creating scientific revolutions – a striking contrast with modern-day American scientists, who seem to want to escape their lab benches for their offices from their first day as assistant professors. Fred was almost completely focused on developing new methods, and the ones he came up with were simple, powerful and elegant in conception. Part of his genius was to keep trying new things, undaunted by failure. When I ran into him years later in the breakfast line at a meeting in Squaw Valley, he asked what I was up to. As I explained a two-dimensional separation method that I had worked out for identifying kethoxal-modified sites in ribosomal RNA, he smiled and asked lots of detailed questions about pH and other experimental conditions. Finally, he said something that could be engraved in stone: ‘I’ll bet you tried a lot of things that didn’t work.’

A highlight of my time in Cambridge was a field trip that everyone took to the Royal Institution in London, to the unveiling of the first crystal structure of an enzyme, lysozyme, which had been solved there by David Phillips and his coworkers. A series of talks on the structure and mechanism was presented in the ancient lecture theatre, with its rows of vertiginously raked seats and oil paintings of legendary British scientists hanging from the walls. All of the giants of Cambridge protein crystallography were seated at the front of the packed hall. But sitting in the centre of the front row was none other than Sir Lawrence Bragg himself, who had created the science of crystallographic structure determination with the structure of sodium chloride more than half a century before, and in whose group at the Cavendish laboratory protein crystallography had been developed, mainly by Perutz and Kendrew. In a time long before computer graphics, the structure was presented with physical models. A CPK model of the polypeptide chain of lysozyme hung straight down from the high ceiling of the auditorium, with its C-terminal residue suspended dramatically an inch above the demonstration table. Phillips then unveiled the structural model, about two feet in diameter. He then turned out the lights and flipped switches that turned lights on for the amino acids in the suspended model – red for hydrophilic, blue for hydrophobic. Next, he turned the lights on in the 3D model, and we all saw the blue lights blink on in the interior and red lights on the surface. The following talks presented explanations of the enzyme’s substrate specificity and mechanism of action. We were stunned.

In Berkeley, I had met the Englishman Tony Tanner, who was on a year’s sabbatical from Cambridge. When he heard that I was headed to the MRC, he urged me to look him up at King’s College, where he was taking a position as head of English studies. When I found him there, he invited us to a sherry party that he was hosting in his rooms the following week. The party was full of students, faculty and other fellows of King’s, holding sophisticated conversations that were far over my head, my 200-word California vocabulary putting me at a serious disadvantage among the elite of one of the world’s greatest intellectual centres. Struggling to feign composure, I was approached by none other than the most intimidating of Cambridge intellectuals: Sydney Brenner. Sydney was one of the founders of the field of molecular biology and was known for his unmatched scientific brilliance as well as his devastating wit. In the MRC cafeteria, he often held forth at a table crowded with Nobel laureates, none of whom were at ease matching wits with Sydney. I held my breath. Sydney looked at me and said, ‘Who are you?’ When I explained that I was a postdoc at the MRC, he asked, ‘What are you working on?’ I told him that we were trying to sequence GPDH, to which he replied, ‘That’s stupid! If you’re a protein chemist, why don’t you work on something interesting – like ribosomes?’

I was speechless. I wanted to crawl under the rug. But during the next days and weeks, I was unable to shake this question. I went to the MRC library and began reading about ribosomes. I began asking everyone about ribosomes. Sydney was right – they were very interesting. And no one seemed to know much about these particles. When I asked who is working on ribosomes, an answer that came up repeatedly was Alfred Tissières, who was starting a group in Geneva. Around this time, Ieuan informed me that he had invited a Hungarian postdoc to come and take my place in the lab. I immediately wrote to Alfred, asking him if he needed a protein chemist. By return mail, he answered, ‘When can you get here?’ We sold our MG YB and bought a Bedford van for a trip to Geneva to meet Alfred. When our landlady Miss Hicks learned that we were headed for the continent, she warned us to ‘be sure to pack a tinned ham’ for the trip; when we reached France, we realised that her concerns were overstated.

And Cambridge followed me to Geneva. Alfred Tissières himself had been a graduate student in Cambridge, and had done his postdoctoral research at Harvard with Jim Watson, who of course had also been a postdoc in Cambridge. The senior postdoc in Alfred’s lab was Rob Traut, who had also been a postdoc in Cambridge, as had Howard Goodman, Jim Dahlberg and Bob Kretsinger. Another ex-Watson postdoc, Peter Moore, did it in reverse, moving to Cambridge from Geneva. After meeting Alfred in Geneva, and learning about what was going on, I joined his group with great enthusiasm and began what became a lifelong fascination with ribosomes. In that fateful conversation at King’s, Sydney Brenner had, in essence, told me what he has told many young scientists: You have but one life and one career; you can spend it working on something boring or something exciting – it’s up to you. Incredibly, it had never occurred to me that it was my choice. I have Cambridge, and especially Sydney, to thank for not pulling punches.

In the end, the crystal structure of GPDH wasn’t solved in Cambridge. It was finally solved in 1973 at Purdue by Michael Rossmann (who had, of course, been a postdoc in Cambridge), using the X-ray data obtained in Cambridge, together with our sequence of the lobster GPDH. When he looked at the structure, he recognised that the three-dimensional fold of the binding site for the NAD cofactor was nearly identical to that of the lactate dehydrogenase structure that he had previously solved. He had discovered the Rossmann Fold.

As for the ribosome, my training began with a crash course from the other postdocs in Alfred’s group, many of whom, including Ray Gesteland, Gary Gussin and Peter Moore, had become seasoned experts during their graduate work in Jim Watson’s lab at Harvard, and were generously and patiently willing to share their wisdom with me. After two years in Geneva, I joined the faculty as an assistant professor at the University of California at Santa Cruz, where I began by attempting to study the mechanisms of action of ribosomal proteins. Our repeated failure to uncover any evidence for ribosomal protein function in translation led to our conviction that the mechanism of translation was based on ribosomal RNA, supported by kethoxal modification experiments that we did in 1972. At the time, this proposal was received by the biochemistry community as a ‘crackpot idea’. Identification of the sites of kethoxal modification led to sequencing the rRNAs, and determining (in collaboration with Carl Woese) their secondary structures. Meanwhile, we developed rapid methods for ‘footprinting’ the binding sites for tRNA, antibiotics, translation factors and ribosomal proteins on 16S and 23S rRNA. These results enabled molecular characterisation of the functional sites of the ribosome, including the A, P and E sites, the decoding site and the peptidyl transferase centre and predicted that their structures all contained universally conserved elements of ribosomal RNA. Footprinting experiments by my graduate student Danesh Moazed led to the discovery of the hybrid states model for translocation. Finally, with the arrival of Marat and Gulnara Yusupov and Jamie Cate, we became involved in ribosome crystallography, obtaining the first X-ray structure of the entire 70S ribosome, complete with mRNA and tRNAs bound to the A, P and E sites. The challenge now facing us is to understand how the molecular dynamics of this complex molecular machine enable its functions. After more than four decades, the ribosome has lost none of its excitement.

CHAPTER 14

At the Laboratory of Molecular Biology: 1965–8

John Abelson

Department of Biology, California Institute of Technology, Pasadena, CA

I arrived at LMB in the autumn of 1965. I had been a graduate student in the department of biophysics at Johns Hopkins from 1960–65. The department of biophysics was mainly a physiology group, a different conception of biophysics than the biophysics of Bragg et al in England. But by the time I came they had added an X-ray crystallographer, Warner Love; an electron microscopist, Michael Beer; and a molecular biologist, Charlie Thomas. I worked with Thomas, who had trained with Paul Doty at Harvard. Our lab worked on the topology of phage and bacterial DNA molecules. Charlie had previously shown that the DNA of bacteriophage T4 was a single linear but circularly permuted DNA molecule but there was controversy as to whether the polynucleotide chains were intact or contained interruptions or gaps. Another physical chemist, David Freifelder, had published evidence that the polynucleotide chains of T4 were not intact and contained nicks. My thesis was to investigate this problem in a number of different phages including T4, T5, P22 and lambda. A number of physical chemical techniques had been applied to this problem, e.g. viscosity, light scattering and analytical centrifugation in the huge Beckman Model E centrifuge. But by the time I had finished we had come to the conclusion that there were only two reliable ways to determine the molecular weight of DNA and these were by electron microscopy where one simply measured the length, and by sucrose density sedimentation. The question of the nicks could be solved by doing alkaline sucrose density sedimentation. By 1965 I had determined that indeed T5 DNA did have nicks and that they were at specific locations. Different variants of T5 had nicks in different places. T4, P22 and lambda did not have any nicks.

I gave a talk at the phage meetings at Cold Spring Harbor that autumn and had my audience with our guru in this field, Al Hershey. He liked my experiments so I wrote up my thesis. I submitted my paper to the Journal of Molecular Biology. This was the place to publish and my reading of the journal had led me to conclude that there was only one place I wanted to go for a postdoc and that was the MRC, to work with Sydney Brenner. Charlie wrote a one-line letter endorsing my application and Sydney accepted me. Then there was a frantic hurry to write my thesis, defend it and make it to Cambridge in time for my wife to begin as a graduate student in history. I passed the non-trivial thesis defence (the two guys before me had failed) and the next day we left from Dulles with our two-year-old daughter Sara and on a cool, bright autumn morning, we rode through London to King’s Cross and made our way to Cambridge.

We had already rented a house in Drosier Road, near the Catholic church, so the next day I made my appearance in the lab. I was cordially greeted by Sydney and Francis and was taken around to meet the students and postdocs. One of the first people I talked to was Roger Freedman, a student of Sydney’s. I breathlessly told Roger about my thesis work and he said: ‘Why would you want to do something like that?’ I found this depressing and although I would leave Drosier Road in the morning to go to work, for a week or so I only walked over to Mill Road and spent the day watching foreign movies at a cinema. But in retrospect, Roger was right (even if obnoxious). How important really was the question of the polynucleotide chains? There were much bigger questions to answer then, and in fact no one has ever returned to this question again. I had come to the right place to ask and to answer big questions.

Eventually I talked with Sydney about a project. By 1965 the code had been solved (not at Cambridge, to the chagrin of Sydney and Francis, but of all places at the NIH by of all people Marshall Nirenberg). But Sydney had beautifully deduced by elegant T4 genetics the sequences of the three stop codons: UAA, UAG and UGA. When a sense codon is mutated to one of the three stop codons the growing polypeptide chain is terminated so stop mutations are, in essential genes, lethal. A number of people, including Sydney, Alan Garen and Dick Epstein, had isolated suppressors to nonsense mutants in E. coli. Suppressors of mutants in which a sense codon had changed to UAG were called amber suppressors (said to be because Epstein’s student was named Bernstein, the German word for amber), to UAA ochre suppressors and to UGA opal suppressors. Each suppressor resulted in the insertion of a characteristic amino acid. For the amber suppressors Su+I inserted serine instead of stop, su+II glutamine and su+III tyrosine. What were these suppressor genes? The most likely model was that they were tRNA genes with mutated anticodons so that the tRNA recognised the stop codon and competed with the chain termination mechanism (at the time itself not known).

Figure 14.1. Nonsense Suppressors.

Figure 14.2. Increase of tyrosine tRNA and suppressor activity in E.coli CA274 su- lac- amber following infection by φ80dSu+III.

I enthusiastically joined this project. My coworkers were John Smith, a senior biochemist in our lab, and in the end four other American postdocs – Art Landy, Howard Goodman, Malcolm Gefter and Dick Russell – and a Czech postdoc, Stan Zadrazil.

Sydney had already prepared the genetic tools for executing this project. In the E. coli genome, the Su+ III gene is near the attachment site for the lysogenic phage φ80. This allowed the isolation of the defective transducing phage φ80dSu+III in which genes near the attachment site are incorporated into the phage genome replacing phage genes. In an early experiment I showed that when a lac amber mutant strain of E. coli is infected with φ80dSu+III, there is an increase in beta-galactosidase activity due to the introduction of the suppressor and a tenfold increase in the relative accepting activity of tyrosine (but of no other amino acid), suggesting that in the infected cell there had been an amplification of a suppressor and a tyrosine tRNA gene.

Figure 14.3. Fractionation of φ80dSu+I tRNA by chromatography on DEAE Sephadex.

But to prove the mechanism of amber suppression suggested above, we needed to show that in a Su+III strain there had been a change in the sequence of a tyrosine tRNA. That is, we needed to sequence the tRNAs. At that time, the sequences of three tRNAs, all from yeast, had been determined; a tRNAAla and a tRNATyr by Robert Holley at Cornell and a tRNASer by Hans Zachau in Germany. These were the first sequences of any nucleic acid. To do this they started with many grams of tRNA and purified the tRNAs by counter current exchange. But upstairs at the MRC Fred Sanger was developing a method for RNA sequencing based on electrophoretic separation of oligonucleotides by two-dimensional electrophoresis. In this method a T1 or pancreatic RNase digest of 32P RNA in about 5 µl is spotted on to a strip of cellulose acetate soaked with a pH 3.5 buffer and electrophoresed at 3–4,000 volts in a large tank of Varsol coolant. This step draws very little current. Then the oligonucleotides are blotted onto a sheet of DEAE paper. The paper is soaked with 7 per cent formic acid and again electrophoresed drawing a very high current. The oligonucleotides are separated by base composition in the first dimension and by size in the second dimension. (This technique is inherently dangerous, and later in La Jolla we had a fire in an electrophoresis tank that destroyed the lab.) Using this method, George Brownlee had made some progress in determining the sequence of E. coli 5S RNA. This was certainly a preferable approach for us because in φ80dSu+III infected cells we showed that when 32PO4 is added to phage infected cells there is a preferential incorporation of 32P into RNA eluting with tRNATyr in column chromatography on DEAE Sephadex.

After another chromatographic step on benzoylated, naphthoylated DEAE cellulose, the 32P tyrosine tRNA was relatively pure and we obtained simple fingerprints, an indication that we could in principle solve the sequence. Here is the T1 ribonuclease fingerprint of the Su+ tRNA.

Figure 14.4. T1 Ribonuclease fingerprint of the Su+ tRNA.

In about a year and with a lot of help from the Sanger lab we solved the sequence of both the Su+ and Su– tRNAs. The T1 spot that moves slowest on DEAE has the sequence: ACUC𝚿 AA*AUCUG where C𝚿A is the anticodon pairing with UAG. In the Su– tRNA the C is a G or a modified G, the anticodon G𝚿A, recognising the tyrosine codons UAU and UAC. So the prediction of the model is correct. Amber suppressors arise from a mutation in the anticodon of a tRNA.

This was the fourth tRNA to be sequenced and it is, I believe, the first time that anyone determined the sequence of a mutational change at the nucleic acid level.

It is interesting to add a word about the use of radioactivity in this work. In order to solve the structure one needed a lot of counts because each T1 product had to be eluted and further digested to determine its sequence. So we used a lot of radioactivity in these experiments. I once did an experiment on a water bath on my lab bench on the second floor in which I inoculated each of 25 individual phage infected cultures in bubbler tubes with 20 mCi of 32P, a 500 mCi experiment! By contrast today a 100 mCi experiment is considered a big deal. There were inevitable spills but we simply did our best to clean them up (and we did not mention them to anyone). However, the biggest mess was made by Brian Clark who also set out to sequence a tRNA by the Sanger method. Brian decided to purify the tRNA by counter current distribution, the method Holley had used. A counter current machine is a huge piece of glassware with a lot of joints, and each one leaked, so the spill onto the floor in that lab was large. As I remember that space was simply cordoned off. Fortunately 32P decays with a half-life of fourteen days.

Figure 14.5. Tyrosine tRNA E.Coli.

But Sydney had much more in mind with this project. Once you have mutated a tRNA gene to become a suppressor, you should be able, in principle, to isolate suppressor minus mutants of the gene that are not the reverse mutation but mutations that impair the function of the tRNA. That is, you could do tRNA genetics. To do this Sydney employed a genetic trick involving the use of a strain he constructed that contained a gal kinase amber mutation, a gal epimerase mutant and the su+III gene. When this strain is grown on a mixture of galactose and glycerol, the suppression of the gal kinase amber allows the accumulation of UDP galactose because of the epimerase mutant. UDP galactose is toxic and kills the cells, but rare suppressor negative mutants occur and these grow on glycerol. At first this method mysteriously gave only exact revertants of Su+III. Dick Russell sorted this out with a brilliant piece of bacterial genetics. In the E. coli genome, it turns out, the tyrosine su+III gene is in tandem with another identical tRNATyr gene so that the most common mutation is a recombination that deletes one of the tRNA genes. Once this had been sorted out we started with strains containing only a single tRNA gene at that locus and those gave interesting mutants, all of which we sequenced.

Figure 14.6. Nucleotide sequence of the su+III tyrosine suppressor tRNA.

It was at this point that I left Cambridge in the autumn of 1968 to go to the department of chemistry at University of California at San Diego in La Jolla, California. After I left the technique of acrylamide gel electrophoresis for the separation of nucleic acids appeared. Sidney Altman, a new American postdoc, and John Smith used this technique to look at some of the mutants and it turned out that the mutation G15 → A accumulated a precursor tRNA, longer by 60 nucleotides at the 5′ end, and further that E. coli extracts contain an enzyme that matures the RNA (RNase P). This was one of the first discoveries of RNA processing. In my lab at La Jolla we were finding the same thing with tRNAs that are encoded in the genome of bacteriophage T4, and others found that ribosomal RNAs are synthesised as precursors and are then matured by a variety of processing enzymes. The field of RNA processing was born. At first there were just a few labs working in this field. But as time has gone by, and many mechanisms of RNA processing, for example RNA splicing, have been discovered, many people have joined the field. The early members of the field formed a society, The RNA Society. It has a journal and an annual meeting and more than 1000 people now come to the meeting. I continued to work in the field and our lab, in 1985, discovered that pre-mRNA is matured by splicing on a large supramolecular complex, that we called the ‘spliceosome’. I am still working on the spliceosome. It contains five different RNAs and more than 100 proteins. To solve the structure of this is a great challenge for the future.

So Cambridge gave me a great start in the right direction. From Sydney I learned that you should always pick an important problem to work on and you should always do it in a genetically tractable system (e.g. C. elegans). From Fred I learned that you can’t begin to understand the problem until you know the sequence. So that’s a pretty simple take-home lesson; do genetics in a system where you know the sequence. Obvious now (or even passé since we know all the sequences). It wasn’t obvious to everyone 45 years ago.

But Cambridge turned out to be important in another way. In science, I have found a community and lifelong friends. Many of these friends were at Cambridge with me. They include Jim Dahlberg, Harry Noller, Tom and Joan Steitz, Glauco Tocchini-Valentini and David Hirsh. But some are no longer with us and in particular, the untimely death of Paul Sigler was a tragedy for us all. So perhaps it is right to end with a photo from the famous lab Christmas party where we did a ballet of protein synthesis. Paul was magic magnesium and I was the 30S ribosome. Paul carried a sparkler and was crucial for the formation of every peptide bond. That’s the way we’ll remember Paul.

Figure 14.7. John Abelson in the ‘ballet of protein synthesis’ as the 30s ribosomal subunit, and on the far right, Paul Sigler as magic magnesium in the LMB Christmas pantomime, 1967. Photo by John Haselgrove.

REFERENCES

J.N. Abelson and C.A. Thomas, Jr., 1966. The anatomy of the T5 bacteriophage DNA molecule. Journal of Molecular Biology 18, 262–91.

J.D. Smith, J.N. Abelson, B.F.C. Clark, H.M. Goodman and S. Brenner, 1966. Studies of amber suppressor tRNA. Cold Spring Harbor Symposia on Quantitative Biology 31, 479.

A. Landy, J.N. Abelson, H.M. Goodman and J.D. Smith, 1967. The specific hybridization of tyrosine transfer ribonucleic acids with DNA from a transducing bacteriophage 80 carrying the amber suppressor gene suIII. Journal of Molecular Biology 457.

H.M. Goodman, J.N. Abelson, A. Landy, S. Brenner and J.D. Smith, 1968. Amber suppression: A nucleotide change in the anticodon of a tyrosine transfer RNA. Nature 217, 1019.

J.N. Abelson, L. Barnett, S. Brenner, M. Gefter, A. Landy, R. Russell and J.D. Smith, 1969. Mutant tyrosine transfer ribonucleic acids. FEBS Letters 3, 1–4.

H.M. Goodman, J.N. Abelson, A. Landy, S. Zadrazil and J.D. Smith, 1970. The nucleotide sequences of tyrosine transfer RNAs of Escherichia coli. European Journal of Biochemistry 13, 461.

J.N. Abelson, M.L. Gefter, L. Barnett, A. Landy, R.L. Russell and J.D. Smith, 1970. Mutant tyrosine transfer ribonucleic acids. Journal of Molecular Biology 47, 15.

R.L. Russell, J.N. Abelson, A. Landy, M.L. Gefter, S. Brenner and J.D. Smith, 1970. Duplicate genes for tyrosine transfer RNA in Escherichia coli. Journal of Molecular Biology 47, 1.

CHAPTER 15

A Three-Dimensional End to a Two-Dimensional Beginning (1965–70)

David DeRosier

Department of Biology, Brandeis University, Waltham, MA

Figure 15.1. David DeRosier at a meeting in honour of Aaron Klug. 9 September 1996, Peterhouse, Cambridge.

Figure 15.2. Joyce Baldwin, David DeRosier and Tony Crowther at a meeting in honour of Aaron Klug. 9 September 1996, Peterhouse, Cambridge.

I arrived in Cambridge in the summer of 1965 with a one-and-a-half-year-old daughter, a pregnant wife, a reservation at the University Arms Hotel on Parker’s Piece, and not a clue about life in England or about the MRC-LMB. I did know that people drove on the wrong side of the road but did not grasp that when crossing the road, I had to look to the right, not to the left. That my daughter and I survived is thanks to the observant British driving public. We took up temporary residence in the University Arms, whose clientele were an older, staid class of people. Our first evening in the hotel’s dining room was a nightmare. The other diners gave us restrained sideways glances as our daughter banged out a tattoo with her feet against a wooden high chair. After that, we fed our daughter early in the room and left her with the maid with whom she helped turn down beds while my wife and I enjoyed a comfortable dinner in peace.

Our first order of business was to find a place to rent. Finding a suitable place was easy. It was the house of Vernon Ingram, who left the MRC-LMB for a year’s sabbatical at MIT and never returned. The difficulty in renting Ingram’s house lay in getting his permission. The estate agent said he would handle the matter. Days went by, money slipped through our hands into the hotel’s cash register, and we heard nothing. I stopped by the estate agent’s office and inquired about the permission, asking if he had an estimate as to when he would hear from Ingram. The estate agent said that the letter had been posted but since it took days to get to the US and more days to receive a reply, he expected it would be at least another week. I asked him if he could phone Dr Ingram. He said he was not authorised to make such expenditures. I told him I would gladly pay for the call, figuring that any transatlantic phone call would be cheaper than another week at the hotel. I know I was a bit brash and I forget how things got sorted out, but they did, and at last the agent took us to the house. He said we had some paperwork to finish and gave us a list of all (and I mean all) the items in the house. The list was in British English, meaning that some names were not familiar. We checked off what we knew, like knife, butter dish or table. By elimination, we tried to deduce the rest. This was how I learned that ‘slice’ is the British term for spatula. Over the ensuing four years in Cambridge I learned many other wonderful terms like lollipop man (school crossing guard who holds a large round STOP sign on a pole), a load of old cobblers (a statement that is rubbish), codswallop (nonsense), Bob’s your uncle (there you are), two bob (two shillings), and knicker (one pound sterling). The new-to-my-ears expressions were a delight, which brings me to my other delight – the British monetary system of the day: guineas, pounds, shillings and pence. One of my lasting memories is teaching the British money system to my relatives. We told them that a guinea was one pound and one shilling or 21 shillings. We said that at an upscale store, prices were often in guineas, which sounded more posh. At a slightly less upscale store, the price of the same item would be in pounds and shillings, whereas at a bargain store the price would be all in shillings, which sounded cheaper. After spending an hour showing them handfuls of the relevant coins and paper money, I decided to give my relatives their final exam. ‘Suppose,’ I said, ‘you purchased an item that cost two and a half guineas and you gave the merchant a five pound note. How much change would you get?’ Even my father-in-law, who was an engineer by training, could not figure out the answer, even with a pencil and paper and given half an hour. The correct answer is left to the reader as an exercise. I have since returned to England and I miss the old coinage.

The Ingram house with its garden of flowers and fenced-in back yard was ideal. We had a coal-fired furnace with central heating. The floor plan was an American-style open plan. There was a doorway and a pass-through from the kitchen to the dining room. The living room and dining room formed an open L. The kitchen had a small pantry open to the outside air. There was an unheated half bath next to the entryway. Upstairs were three bedrooms and a full, heated bath. We bought a TV and a TV licence, and I learned still more Briticisms watching the telly. One of my favourite shows was Till Death Us Do Part, an irreverent comedy about the working class. I found amusement with new phrases such as ‘scarse git’, a frequently used insult on this show. I also delighted in British surnames that echoed the occupation of the owner. Where else in the world would we have hired a plumber named Mr Waters or had a gynaecologist called Miss Bottomley? My wife, with the help of a midwife and oversight by Miss Bottomley, delivered our son at the Evelyn Nursing Home on Trumpington Road. The delivery went smoothly while I sat in the waiting room. I left in the evening to return to the Ingram house and take over from our babysitter. As I was leaving the Evelyn, the staff were removing the plants from my wife’s room. The reason, I learned, was that although plants generate oxygen during the day, they produce carbon dioxide at night and must be removed. I made a note never to spend a night in a forest.

The new car I had ordered was not yet ready for delivery, although it should have been. So I took the bus to the lab to meet Aaron Klug, my mentor. We chatted briefly and he assigned me to an office across the hall. He then disappeared on his bike to tutor his Peterhouse students. I sat in my office, which I shared with three others. No one was in, so I got up to look around the lab. On my way out the door, I noticed that the brass name holder over the door had a loose screw and hung askew. I paused to use my thumbnail as a screwdriver to fix the holder. About that time, a shortish man with a somewhat rumpled sweater under a tweed jacket came along the hall and stopped to look at me. I thought that he was part of maintenance about to suggest I leave the askew name plate alone, but I held my tongue from asking who he was or what he did. He paused where I stood and then said: ‘Perhaps I should introduce myself. I am Max Perutz.’ I introduced myself, glad that I had held my tongue.

Now that I had met Dr Perutz, I had no idea how to address him when next we met. At my alma mater, the University of Chicago, all our professors, even my PhD advisor, were addressed as doctor. Surely, in the more formal UK, one would use doctor or professor. I continued on down the hall and wandered into a lab where technicians were making liquid media. I introduced myself. They seemed very friendly and so I asked, ‘How does one address Dr Perutz?’ One of the women replied, ‘You mean Max?’ Aha, I thought, the atmosphere is informal; everyone from the technicians to the director worked on a first-name basis. I mention this because I came to realise that it was an important aspect of the lab in that it encouraged students and postdocs to talk freely with its senior members.

Many of my important, research-altering talks with lab members took place at coffee (elevenses), lunch at 1, and tea at 4. Many institutions have tried to capture this atmosphere in order to promote the same high level of scientific chat as that found at the MRC-LMB. I am not sure that any other place has succeeded. Perhaps the key is not just the canteen but more importantly the people who ate and drank there. I certainly benefited from the three daily treks for these breaks. Some days I sat with the folks in the molecular biology division. Sitting at the same table as Francis Crick and Sydney Brenner was always a treat. Sometimes I would sit with the people in the structural division. I particularly remember that John Finch, Aaron and I would often sit together at tea or coffee having a serious discussion and that Aaron would want to write down an equation or some thought about an experiment to try. He needed something to write on. John often liked to have a KitKat bar, and Aaron would then use the KitKat wrapper as his note pad. Then he would admonish me to keep this important document. In my filing cabinet, I had a folder labelled ‘Aaron’s Scraps of Paper’. In addition to the KitKat wrappers, it also contained scraps of paper napkins in various shapes and sizes.

A couple of years ago I wrote a chapter for Methods in Enzymology (2010), in which I gave the history of the development of image analysis and 3D reconstruction as I remembered it. I did not want to go over again the detailed story of how 3D reconstruction from electron micrographs came into being, so I thought I would say why I thought these developments came to pass at this time and in this lab. The first element that led to the development of 3D reconstruction was the development of optical filtering, a technique on which Aaron and I worked that extracted the periodic features from an electron micrograph of a biological structure. Often, the images of interesting periodic structures such as the helical tobacco mosaic virus (TMV) were confused because in addition to noise, the images suffered from the superposition of both the front and the back sides of the structure. (These two-sided micrographs reminded me of looking at medical chest X-rays where the ribs from the front and back crisscross in a confusing pattern.) We found we could use filtering to extract the image of just one side free of noise and of the other side. In our first attempt we filtered an electron micrograph of TMV. In the filtered image we could clearly see the protein subunits of TMV as a beautiful array of small blobs. Our excitement was tempered by the knowledge that our beautiful image was still two-dimensional, not a three-dimensional image. While we could subtract one side from the two-sided image, we could not by this method see the internal features of the extracted side. These internal features were superposed in the filtered image. Given the unusual properties of helical structures and their Fourier transforms, however, Aaron and I realised we could generate a 3D map of a helical structure from a single electron micrograph. To do so we needed to measure the amplitudes and phases of the diffraction patterns seen in our optical diffractometer. Since we had experience with our optical diffractometer/filtering apparatus, we decided to measure the phases optically using holography. Quickly, Dave Hart working in the machine shop, headed by Len Hayward, made modifications to our optical apparatus.

Yogi Berra, an American sports figure known for his amusing remarks, known as Yogi-isms, is reputed to have said: ‘In theory, there is no difference between theory and practice. In practice, there is.’ In theory, holograms preserve phase information, which is easily extracted by measuring the positions of fine fringes in the hologram. In practice, it was not easy. It was a headache-inducing, madness-fomenting, mouth-frothing nightmare. To sum up, it was ghastly, and I wished for a canteen that served alcohol instead of tea. The holographic fringes carrying the phase information were so fine that I needed a high magnification micrometer to make the measurements. After several weeks of agony, I did get our first set of phases from John Finch’s best micrograph and using Ken Holmes’ FORTRAN program for helical Fourier data, I generated the first 3D reconstruction. We now had a 3D map of the helical tail of the T4 bacteriophage. There was a good deal of excitement. Even Francis Crick stopped by to look at the map.

3D reconstruction had almost been born. I said almost because it was clear to me, who had to make the measurements, that the technique of holography was not the solution to getting phases. I did not think I would be able to measure phases on even one more micrograph. Which brings us to the second element: the flying spot densitometer developed by Uli Arndt with the help of Paul Phizackerley. The mechanical parts were built by Ernie Norman in the LMB, the electronic parts soldered together by Frank Mallett, and the software worked out and put on punched paper tape by Tony Crowther. The instrument, which became available in 1966, seemed the answer to the phase problem. It was built to measure X-ray crystallographic intensities, which have their own phase problem. We wanted to measure the densities on our electron micrographs and compute phases. Tony Crowther helped digitise our images.

The third elements needed were the computers, not only the one that ran the flying spot densitometer but also the IBM computer in London that ran data analysis. The computer (Argus) that operated the densitometer also ran the X-ray diffractometers. Programs were written in the octal codes for machine instructions. Programs were read in on paper tape and data were put out on large reel magnetic tapes. Computer programs for data analysis were written in FORTRAN on punch cards for the IBM 7090 in London. I spoke to the crystallographers about using their FORTRAN programs to calculate phases. After all, if I put in densities (with phase angles of zero) instead of amplitudes and phases, I would get a Fourier transform. That was in theory, but in practice – looking at their algorithms – it was not possible. Instead, with much help from Tony Crowther and later in collaboration with Peter Moore, I taught myself FORTRAN and began to put together a series of programs to analyse images digitally. Because computing using FORTRAN programs was already in place, it was a relatively simple matter to get processing under way given a FORTRAN program and magnetic tapes with data. Here’s how a day of computing went: One of the computer assistants would take everyone’s punch cards and magnetic tapes into London in the late morning and bring back the output in the afternoon. There was then a mad scramble to figure out what was right and what was wrong with the morning’s computations, to make changes, and to get one’s cards and tapes ready for the late afternoon run to London. Everything worked well and we soon were able to compute amplitudes and phases and generate 3D maps of helically symmetric structures.

The fourth element that led to the development of a general algorithm for 3D reconstruction was a project that John Finch (who took all the electron micrographs) and Aaron had been working on for several years. They had entered a controversy about human wart virus, a spherical virus. One faction said that the virus had 42 capsomers. (Capsomers are clusters of protein subunits seen as morphological features in the electron microscope.) The other faction insisted the number was 92. Both used evidence arising from electron micrographs. The former faction counted capsomers in puddles, which they argued came from viruses that had fallen apart. The latter faction counted morphological features on the surface of intact viruses, although the confusion of detail from the superposition of the near and far sides made counting tricky. Aaron and John wrote a short article saying that both groups were wrong and the number was 72. (The theory of quasiequivalent virus shells proposed by Don Caspar and Aaron only allowed values of 12, 32, 42, 72, 92 and higher for the numbers of capsomers.) John and Aaron had found a few one-sided images and showed that the pattern of capsomers on this virus fit a 72- but not a 92- nor 42- capsomer structure. John and Aaron had clearly analysed the one-sided images correctly but rather than ending the controversy, it made things worse. Aaron and John needed to do more. The two of them realised that because the one-sided images were only a small fraction of all the images, they had to show that their 72-capsomer model could account for all or almost all the images. John and Aaron used their model to simulate two-sided images and showed that they could account for essentially all two-sided images in great detail.

Some of the opposing researchers still disagreed, producing two-sided images that sort of but not exactly looked like the images seen in the electron microscope. The details were not correct but this did not bother them because the science of interpreting images had been up to that point a matter of interpretation. Aaron and John supposed that the other factions might be convinced by tilting experiments because two different models must behave differently when tilted. John and Aaron produced pairs of micrographs in which the very same field of virus particles were tilted by a few degrees between exposures onto film. They then compared their tilt-pair micrographs to simulated images in which their model had been tilted about the same axis and by the same tilt angle as the viruses. The model clearly explained the changes seen in the images, and finally the controversy seemed to end.

The deeper question, however, was this: How many tilts are needed to prove one has a unique model, and how accurate do the features of the model have to be? Aaron and I saw how reconstructions of the structure were possible for helical structures with their special symmetry, but we could not see immediately how to adapt the method to non-helical symmetries. The impediment was in the choice of words to describe the images. Images of structures seen in the electron microscope were called two-sided superpositions and not projections. Even though both words mean essentially the same thing, the latter immediately leads to thoughts of the relationship between the Fourier transform of a projection and the Fourier transform of the 3D object that gave rise to the projection. The Fourier transform of a projection is a 2D slice through the 3D Fourier transform of the 3D structure. Once that connection is made, the rest of 3D reconstruction becomes obvious. One can build up the 3D Fourier transform by collecting the requisite number of 2D slices.

This theory of 3D reconstruction was very satisfying because it answered the two questions in the previous paragraph. It told us how many views one needed to produce a 3D image or map to a given resolution. This is not the place to provide that mathematical answer, but rather to say that the theory also obviated the need to think up models and test them by tilting and projecting for comparison to tilt pairs. Indeed one can instead generate a 3D map directly from the electron micrographs.

The fifth element that made possible the development and implementation of 3D reconstruction were my colleagues from whom I got so much. I enjoyed learning from and arguing with Aaron. He taught me about helical diffraction, optics and alignment of optical systems using the Boys points, about holograms, about Fresnel images and Moiré patterns, and much more. There was seldom a topic I brought to Aaron that he had not thought about, read about, and made notes which he kept in his filing cabinet. Unlike Max, who mounted his own crystals and collected his own X-ray data, Aaron himself did not collect data, at least not while I was there, but he wanted to hear in detail exactly what physical manipulations were involved and what things looked like. He wanted to imagine doing the experiment himself. Sometimes, though, he did join me at the optical bench where we amused and educated ourselves by making Fresnel images of periodic objects, for example. One of the things Aaron delighted in was showing guests our optical filtering experiment. We would put our favorite periodic object (a T4 polyhead) into the apparatus and have the visitor look at the unfiltered image, which was a mess of noise and superposition. We would then pop in the filter mask while the visitor continued to look at the image and in less than a second the image was transformed into a beautiful hexagonal array of protein subunits. Aaron and I occasionally discussed or argued mostly about things one of us said that were misunderstood by the other. Sometimes we disagreed about substantive things. I can’t remember any of the topics but I do know that in the end, we usually came to an agreement with one or the other of us yielding. When I left the LMB, I thought I still had a lot I could learn from Aaron.

John Finch gave me my initial insights into symmetry and the techniques of electron microscopy and he provided all the micrographs we analysed. Ken Holmes gave me his general FORTRAN program for generating 3D maps from layer line data and a good deal of advice that I greatly appreciated. I learned a great deal about the solving of simultaneous equations from Bob Diamond, who was developing algorithms for fitting peptide chains to 3D density maps. I am especially indebted to Tony Crowther, who showed me the point of inverting matrices by calculating eigen values and eigen functions. He also was very important in my effort to learn FORTRAN programming. Tony and I also had many discussions about other topics like non-crystallographic symmetry. From Francis Crick, I learned how to think about the correctness and implications of equations without having to write down any mathematics. I also had the good luck to spend an hour with W.L. Bragg who stopped in that lab one day while Aaron was away. I was put in charge of telling Bragg about 3D reconstruction. The night before, I brushed up on the equations, making sure the πs were where they should be and the signs were correct. When I began to speak to him while writing equations on a tiny chalkboard, he stopped me and indicated that he did not want equations but rather for me to tell him in words what the equations said. I thank Francis Crick and Aaron, who taught me to put equations into English, for giving me the confidence to tell someone as notable as Bragg about the use of Fourier transforms to carry out 3D reconstruction.

The MRC-LMB had all of the elements I have described in place for reasons unrelated to the development of 3D reconstruction. I have since learned that the ideas for its development were being thought about in other labs such as those of W. Hoppe, M. Moody and R. Hart, but it was the very fertile environment of the lab that made its development and implementation possible.

After four exciting years, I decided to head back to the US to start my own lab, but to which of the many interesting biological structures should I apply 3D reconstruction? Aaron suggested we ask Sydney Brenner what I should do when I returned to the United States. What I had hoped for was some insight into an interesting biological structure I might investigate. Instead Sydney said: ‘I know exactly what David should do. Have a pizza.’

REFERENCES

DeRosier, D.J., 2010. 3D reconstruction from electron micrograph: A personal account of its development. Methods in Enzymology 481, 1–24.

DeRosier, D.J. and A. Klug, 1968. Reconstruction of three-dimensional structures from electron micrographs. Nature 217, 130–4.

CHAPTER 16

My Formative Years as a Postdoc at the Laboratory of Molecular Biology (1966–8)

Harvey Lodish

Whitehead Institute for Biomedical Research

Departments of Biology and Bioengineering, MIT

INTRODUCTION: THE MECHANISM AND REGULATION OF POLYPEPTIDE CHAIN INITIATION

1966 was an important year in molecular biology, as it saw the completion of the genetic code and the beginnings of an understanding of the molecular regulation of gene expression. As an example, the 1966 Cold Spring Harbor Symposium ‘The Genetic Code’ brought together all of the leaders in molecular biology. It was my first major international meeting. I remember listening with rapt attention to a large number of talks on nonsense codons and their polar effects on expression of downstream genes in a polygenic operon and on the role of specific tRNAs in suppressing these mutations. Individual tRNAs had just been purified and were beginning to be analysed, and interactions of tRNAs with aminoacyl tRNA synthetases and with ribosomes were just beginning to be characterised. All of this presaged many exciting discoveries in subsequent years concerning the molecular details of polypeptide chain elongation and termination.

But for me the most exciting developments concerned polypeptide chain initiation and its regulation. Bob Webster in Norton Zinder’s lab had shown that the f2 coat protein, synthesised in an E. coli cell free system, had at its N-terminus a formyl-methionine residue preceding the normal N-terminal alanine residue. Contemporaneously, Gary Gussin and Mario Capecchi in Jim Watson’s lab identified N-formyl methionyl tRNA and showed that it was the tRNA used for initiation of bacterial protein synthesis. Many papers at the Cold Spring Harbor meeting focused on characterisation of the bacterial initiator methionyl tRNA, the linkage of methionine to the tRNA by the methionine aminoacyl tRNA synthetase, and the subsequent addition of the N-formyl group to the methionine.

I arrived at the LMB in July 1966 with a fellowship from the American Cancer Society, a pregnant wife, a complete ignorance of foreign cultures, and only a vague idea of the projects I wanted to carry out concerning the regulation of polypeptide chain initiation.

My PhD thesis under Norton Zinder at Rockefeller University had focused on a genetic analysis of the RNA bacteriophage f2, generating and analysing amber (nonsense) and temperature sensitive mutants. I identified mutations in three phage genes – the coat protein, a subunit of the RNA polymerase, and a new ‘maturation’ protein, which was essential for proper assembly of the virus and for attachment to bacterial cells. Joan Steitz, then a PhD student with Jim Watson at Harvard and later a valued colleague and friend at the LMB, independently identified the ‘maturation’ protein in a related RNA phage. I also showed that the plus strand viral RNA served as template for synthesis of the complementary ‘minus’ strand by the virus-encoded RNA polymerase, and that the minus strand in turn served as template for the synthesis of abundant daughter ‘plus’ strands.

Translational control of protein synthesis was one of the big questions that excited me, especially the regulation of polypeptide chain initiation in bacteria by N-formyl methionyl tRNA. As I wrote in the introduction to my Nature paper two years later, ‘One way in which an organism can regulate the synthesis of specific proteins is by translation of the different genes on a polygenic messenger RNA at different rates and different times,’1 but there were few systems with which one could study this problem.

MY RESEARCH AT THE LMB ON THE REGULATION OF INITIATION OF BACTERIOPHAGE F2 PROTEIN SYNTHESIS

After talking with many people at the LMB, especially Mark Bretscher, I soon realised that ‘an ideal system for studying this type of regulation in vitro is the single stranded RNA from small bacteriophages such as f2, for it is the only purified polycistronic messenger that makes defined proteins in a cell-free system’.1 With the encouragement of my mentors Sydney Brenner and Francis Crick I embarked on this study, but first I needed an assay to quantify initiation of all three phage-encoded proteins in an E. coli cell-free system. Since all phage proteins were believed to initiate with N-formyl methionyl tRNA, I used N-formyl [35S] methionyl tRNA to label the N-termini of phage proteins produced in an E. coli cell- free system.

This was not a trivial issue as there was then no commercial source of [35S] methionine and we had to make it ourselves! I learned from Fred Sanger how to do this. Briefly, one put a smidgen of bakers’ yeast in a test tube with about 25 ml of a minimal medium, added 50 mc (millicuries!) of [35S] sulfate, set the culture on the desktop, and bubbled in air until the yeasts grew to confluence. The cells were recovered by centrifugation, washed, boiled, and the extract incubated with a crude mixture of proteases to generate free amino acids. The entire mixture was then streaked in a narrow line near the top of a foot-wide sheet of chromatography paper, then subjected to descending liquid chromatography to resolve the [35S] labelled amino acids and other compounds. The dried paper was exposed to a sheet of X-ray film for about a second to identify the region that contained the desired [35S] methionine, which was then eluted into a small test tube and stored frozen. As I recall, we did not wear gloves or take special precautions when carrying out this procedure since [35S] was a weakly-penetrating beta emitter.

Fortunately one preparation of [35S] methionine allowed me to do several experiments and I remember sharing it with a few lab colleagues. Mark Bretscher, my bench mate and friend, showed me how to make N-formyl [35S] methionyl tRNA to label the N-termini of proteins. Briefly, we incubated unfractionated E. coli tRNA with a crude S100 E. coli extract, [35S] methionine, and formyl tetrahydrofolic acid as a formyl donor, producing a mixture of radiolabelled N-formyl [35S] methionyl initiator tRNA and [35S] methionine attached to the tRNAmet that labelled interior methionine residues. The recovered tRNA was then incubated with another crude cell extract that removed [35S] methionine but not N-formyl [35S] methionine from tRNA, resulting in a radiochemically pure N-formyl [35S] methionyl tRNA.

Next, and again with Mark’s help, I translated purified f2 RNA in an E. coli extract containing the N-formyl [35S] methionyl tRNA, recovered the total protein population, digested it with a mixture of trypsin and chymotrypsin, and resolved all of the peptides by a two-dimensional system – descending paper chromatography followed by paper electrophoresis in the giant gasoline-filled high voltage electrophoresis tanks that were then commonly used at the LMB. Autoradiography revealed only 6 [35S] – labelled peptides. I quickly showed that three contained an N-terminal N-formyl [35S] methionine, and that three were the corresponding peptides from which the blocking N-formyl residue had been removed by an enzyme in the cell extract. Furthermore, the three with an N-terminal N-formyl [35S] methionine corresponded to the N-termini of the three phage proteins – the coat, maturation, and polymerase proteins! Thus I had an assay for analysing and quantifying the rate and extent of initiation of the three cistrons on this polycistronic mRNA.

My experiments indeed generated several insights into translational control. I showed, first, that initiation of translation of the coat and maturation proteins occurred simultaneously and independently of each other, but that much more coat protein was produced. This was among the first indications that different AUG initiation codons on a polygenic RNA initiate protein synthesis at different rates.

Second, I showed that initiation of translation of the phage RNA polymerase gene kinetically followed that of the coat protein. Further, initiation of the polymerase gene actually depended on continued translation of the coat gene, since an amber mutation early in the coat gene not only blocked synthesis of the full-length coat protein, but also reduced the rate of initiation of the polymerase gene. Thus we had replicated in a cell-free translation system the polar effect of an amber mutation on translation of the downstream gene in a polygenic mRNA.

I then hypothesised that ribosome movement along the phage RNA during translation of the coat protein gene somehow ‘opened up’ the RNA secondary structure and exposed the initiator AUG codon of the polymerase gene. Supporting this, I showed that a fragment of the phage RNA that lacked the coat gene initiated translation of much more polymerase protein than did the normal RNA, and that this initiation occurred without a lag period. Apparently deletion of the coat gene indeed ‘opened up’ the RNA secondary structure.

SYDNEY BRENNER AND FRANCIS CRICK

Sydney and Francis were amazing postdoc mentors, but very different in their interactions, attitudes and interests.

Sydney was the consummate experimentalist, analytical and critical, seemingly prowling the lab at all hours of the day and night. As should be evident, Sydney allowed me – really encouraged me – to design my own research agenda and to carry out my own experiments. Indeed all three of the papers I published while at the LMB had only my name in the author list.1–3 But he was constantly discussing experiments with me, encouraging me when I ran into problems, and he would always suggest new experiments and ideas.

Sydney was very supportive of the careers of all of his postdocs. I was offered a faculty position in the biochemistry department at UC Berkeley during the spring of 1966, and signed the actual contract just after I arrived at the LMB. (I remember having some difficulty explaining to a barrister in Cambridge what a loyalty oath was, why California required it, and why it needed to be notarised.) Despite the fact that the biochemistry department was considered number 1 in the United States, for some reason Sydney did not like Berkeley and tried to dissuade me from taking the job.

In any case that autumn Ronald Reagan was elected governor of California and started to put salary freezes and other restrictions on the UC faculty. The reason, it was said, was to get them to focus less on anti-war politics and more on teaching students. In part because of Sydney I was getting very apprehensive about moving to Berkeley and in November, soon after the election, I resigned from the UC Berkeley faculty. Berkeley was not happy but Sydney was. He immediately contacted faculty at Harvard, Yale, University of Chicago and other universities expanding in this new field of ‘molecular biology’, and soon I had several interviews set up. I am proud of the fact that I am at MIT because of both Sydney Brenner and Ronald Reagan.

And I remember when, two years later, Joan and Tom Steitz were having huge difficulties getting tenure-track faculty positions in the same institution. Nepotism rules, preventing spouses from having a position in the same department or university, were still the norm then. Sydney sprang into action and somehow arranged for Yale to offer both of them positions in the same department. The rest, as they say, was history. Importantly, it was unusual then for a male to promote the independent career of a female scientist, but Sydney was very unusual in many ways! I learned from this, and am proud of the fact that I in turn have mentored many female students and fellows, including 23 who had children while in my lab.

Francis was the philosopher and deep thinker. During my first year at the LMB I had little contact with him. But in the summer of 1967 I began developing the notion that RNA secondary structure was playing a key role in the regulation of phage RNA translation, and then Francis became totally fascinated by this apparently novel concept. He and I, and also Sydney and John Smith, spent many hours talking about these ideas and discussing ways in which functionally important RNA secondary structures might have evolved and in turn influenced the evolution of DNA sequences. (Of course, at the time we had no sequence of any gene.)

I learned from both Sydney and Francis how to mentor young scientists, and tried to put this into practice with my own students and postdoctoral fellows. Indeed, years later when I received the Mentoring Award from the American Society of Hematology, I began my speech by acknowledging specific lessons I learned from Sydney and Francis.

NON-SCIENTIFIC LESSONS I LEARNED AT THE LMB

The LMB was a remarkably collegial laboratory, then very small in size but very intense and collaborative. It was probably during my first day there that Max Perutz told me how different the LMB was from American laboratories like those at the Rockefeller. Paraphrasing his words from over 45 years ago, they went something like this. ‘In America there is lots of money for research, and everybody works in the lab as many hours as possible doing as many experiments as one can think of, hoping that one or two will actually give you an interesting result. Here (the LMB and more generally the UK) money is tight so we have to be more efficient. Before you actually do an experiment you discuss it with as many colleagues as possible during morning coffee, over lunch, or during afternoon tea, or ideally during all three. Only then, when you have refined your ideas, do you actually do an experiment. Then you discuss your results at coffee, lunch, and tea for a few days, go back to the lab bench, and do another experiment.’

The point is that this actually worked. I got many good ideas and criticisms from virtually everybody in the lab; I remember in particular long conversations with George Brownlee, Steve Martin, Joan Steitz, Max Perutz, Fred Sanger, and many many more. The only problem was the weight I gained eating all of the rich pastries and cookies over coffee and tea.

I learned a lot about other areas of molecular biology as well. For example, Paul Sigler was finishing the three-dimensional structure of chymotrypsin, building a huge erector set-like model in a small room in the basement. Many evenings I would go there and talk with Paul, David Blow and others; my recollection is that Paul let me add one amino acid (I believe the catalytic aspartate) to the actual chymotrypsin structure. The annual lab retreat, where all of the lab directors spoke, as well as the many seminars, gave me a deep insight into multiple emerging areas of science.

Fifteen years later the LMB served as one of the two ‘model laboratories’ for the new Whitehead Institute; the other was the Salk Institute, where David Baltimore had worked for several years. Our cafeteria was patterned after the canteen at the LMB; we wanted a place where everybody in the building and in the broader MIT biology community could gather during the day over coffee, tea and good food. We wanted the Whitehead, like the LMB, to be greater than the sum of its parts – to encourage the kinds of spontaneous collaborations and interactions that characterised the LMB when I was there.

For me the years at the LMB were also a time of considerable personal growth. Our daughter Heidi was born in October 1966 and our son Martin in June 1968. My wife will tell you that I did not instinctively understand what it meant to be a father and spent altogether too much time in the lab. But I did learn to enjoy the children and to play with them during the few times I was away from the lab, usually most Sundays.

And learning the cultural differences between England and the US was an eye-opener. As an example, backpacks for carrying babies were just coming into vogue in the US and we brought one with us to England. When we visited Windsor Castle with Heidi facing backwards in the backpack and waving to passersby, we were photographed as mini-celebrities; apparently no one had ever seen a child in a backpack and more than a few told us we were abusing her.

Pam and I travelled a lot in England; I got to know Ely Cathedral so well that I would give tours about its history and architecture. We also travelled a lot on the continent, mostly visiting labs, giving lectures and going to museums – again a wonderful introduction to different cultures, people and ways of life.

One legacy is not only that two of our children are British citizens (subjects?), but oddly so are their five children; all have visited the UK and both Heidi and Martin spent many months as adults living and working in England.

MY RESEARCH AS A NEW INDEPENDENT INVESTIGATOR ON THE MECHANISM AND REGULATION OF POLYPEPTIDE CHAIN INITIATION

I left the LMB in July 1968 to become an assistant professor in the biology department at MIT. Whether by accident or design I still do not know, but I was given an office next door to David Baltimore, my old friend from Rockefeller days, and we shared three large research laboratories. David and his postdoc (then wife) Alice Huang introduced me to vesicular stomatitis virus (VSV). One VSV gene, the G or glycoprotein, became invaluable in studies we carried out in the 1970s defining the now familiar endoplasmic reticulum to Golgi to plasma membrane pathway for glycoprotein synthesis.4–6 Later, Flora Katz and Jim Rothman developed in vitro systems where we could translate the VSV G mRNA and show co-translational endoplasmic reticulum membrane insertion and core glycosylation of this transmembrane glycoprotein.7–9 But all this came later.

The immediate questions on which I focused centered on understanding the mechanisms underlying the differential rates of initiation of translation of bacterial and bacteriophage mRNAs. First, together with Hugh Robertson, then at Rockefeller but later a postdoc at the LMB, we showed definitively that the third f2 protein produced in an E. coli cell free system was indeed the maturation protein10 and also that nascent f2 RNAs could direct synthesis of all three phage proteins.11

During this period initiation factors for protein synthesis were being identified and purified, and a major question was whether initiation factors or ribosomes regulated the rates or specificities of polypeptide chain initiation. To address this question I developed a cell-free translation system from Bacillus stearothermophilis. Strikingly, B. stearothermophilis extracts could synthesise the f2 maturation protein but not the two other phage proteins. I went on to show that the specificity was due solely to the 30s ribosome subunit; the source of the 50s subunit or any of the initiation factors made no difference.12,13 I also carried out several other experiments to show that RNA secondary structure indeed restricted initiation of both the f2 maturation protein and RNA polymerase genes; chemical or thermal melting of the RNA increased initiation of translation of these genes manyfold.14,15

But the real question for me during the late 1960s was the mechanism of initiation of translation of mammalian mRNAs, since there was no N-formyl methionyl tRNA in any eukaryotic cell. David Housman, my second graduate student, together with Tom RajBhandary, a new MIT faculty colleague, showed that haemoglobin polypeptide synthesis initiates with an initiator methionyl tRNA that is not formylated. It was known that eukaryotic cells contained two species of methionyl tRNA, and that one of them could be formylated by the E. coli transformylase. David showed that the artificially formylated yeast N-formyl methionyl tRNA indeed initiated synthesis of haemoglobin in a rabbit reticulocyte cell free system, and that the normal initiator methionine, but not the initiator N-formyl methionine, was subsequently removed by a cellular enzyme.16 This led to a long series of studies on the regulation of translation of α and β globin mRNAs, showing (well before globin mRNAs were cloned or could be quantified directly) that reticulocytes had more mRNA encoding the α globin chain than the β but that translation of α mRNAs was initiated less efficiently than the β globin mRNA.17–20 However, the details of these studies would require another essay.

Nonetheless, it should be clear that the scientific and nonscientific lessons I learned at the LMB were indeed formative in getting my independent research career off the ground. I will be forever grateful to my mentors and colleagues there for their friendship, advice and criticism.

REFERENCES

1. H.F. Lodish, 1968. Bacteriophage f2 RNA: control of translation and gene order. Nature 220, 345–50.

2. H.F. Lodish, 1968. Independent translation of the genes of bacteriophage f2 RNA. Journal of Molecular Biology 32, 681–5.

3. H.F. Lodish, 1968. Polar effects of an amber mutation in f2 bacteriophage. Journal of Molecular Biology 32, 47–58.

4. D.M. Knipe, D. Baltimore, H.F. Lodish, 1977. Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus. Journal of Virology 21, 1149–58.

5. D.M. Knipe, D. Baltimore, H.F. Lodish, 1977. Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus. Journal of Virology 21, 1128–39.

6. D.M. Knipe, H.F. Lodish, D. Baltimore, 1977. Localization of two cellular forms of the vesicular stomatitis viral glycoprotein. Journal of Virology 21, 1121–7.

7. F.N. Katz, J.E. Rothman, V.R. Lingappa, G. Blobel, H.F. Lodish, 1977. Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proceedings of the National Academy of Science of the United States of America 74, 3278–82.

8. J.E. Rothman, F.N. Katz, H.F. Lodish, 1978. Glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein. Cell 15, 1447–54.

9. J.E. Rothman, H.F. Lodish, 1977. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature 269, 775–80.

10. H.F. Lodish, H.D. Robertson, 1969. Cell-free synthesis of bacteriophage f2 maturation protein. Journal of Molecular Biology 45, 9–22.

11. H.F. Lodish, H.D. Robertson, 1969. Regulation of in vitro translation of bacteriophage f2 RNA. Cold Spring Harbor Symposia on Quantitative Biology 34, 655–73.

12. H.F. Lodish, 1969. Species specificity of polypeptide chain initiation. Nature 224, 867–70.

13. H.D. Robertson and H.F. Lodish, 1970. Messenger characteristics of nascent bacteriophage RNA. Proceedings of the National Academy of Science of the United States of America 67, 710–16.

14. H.F. Lodish, 1970. Secondary structure of bacteriophage f2 ribonucleic acid and the initiation of in vitro protein biosynthesis. Journal of Molecular Biology 50, 689–702.

15. H.F. Lodish, 1971. Thermal melting of bacteriophage f2 RNA and initiation of synthesis of the maturation protein. Journal of Molecular Biology 56, 627–32.

16. D. Housman, M. Jacobs-Lorena, U.L. Rajbhandary, H.F. Lodish, 1970. Initiation of haemoglobin synthesis by methionyl-tRNA. Nature 227, 913–18.

17. H.F. Lodish, 1971. Alpha and beta globin messenger ribonucleic acid. Different amounts and rates of initiation of translation. Journal of Biological Chemistry 246, 7131–8.

18. H.F. Lodish, 1974. Model for the regulation of mRNA translation applied to haemoglobin synthesis. Nature 251, 385–8.

19. H.F. Lodish and M. Jacobsen, 1972. Regulation of hemoglobin synthesis. Equal rates of translation and termination of α- and β-globin chains. Journal of Biological Chemistry 247, 3622–9.

20. H.F. Lodish and D.G. Nathan, 1972. Regulation of hemoglobin synthesis. Preferential inhibition of and globin synthesis. Journal of Biological Chemistry 247, 7822–9.

CHAPTER 17

Memories of My Time at the MRC-LMB in Cambridge

Jim Dahlberg (with Fred Sanger, 1966–8)

Emeritus Professor, University of Wisconsin-Madison

Interim CEO, Morgridge Institute for Research, Madison, WI

Writing this brief memoir has given me the opportunity to shake loose a few cobwebs and revive some old memories. As is the case with other authors in this collection, my experiences at the MRC (as we called the MRC-LMB at that time), have influenced my life in many ways, both scientifically and personally. What follows are recollections of someone who was fortunate to be a postdoctoral fellow at a very exciting period in the history of that laboratory and the entire field of molecular biology.

FRED

I joined Fred Sanger’s laboratory as a post-doctoral fellow in the autumn of 1966, shortly after receiving my PhD in biochemistry at the University of Chicago. As for many other individuals fortunate enough to spend time with Fred, his demeanour and approach to science greatly influenced me and my own career. I recall several of our conversations with fondness. He would often stroll through the lab and ask ‘What’s up?’, in a very friendly, non-threatening way. It took me a while to learn that the low-key and modest nature of Fred’s advice belied the insight that it contained. Rather than tell me that I was mistaken or doing something wrong, in his soft-spoken manner he would say, ‘You might consider…’.

Fred’s modesty is well recognised. More than once, when I asked him a question about some problem I was having with chemistry, he would answer: ‘Well, I really don’t know much about chemistry.’ Shortly after setting up my own laboratory at the University of Wisconsin in Madison, I was thrilled when Fred asked to come to my lab for a week, to learn how to make α-32P-labeled nucleoside triphosphates; back then, one could not just order them from a company. A few years later, I returned to Fred’s lab, along with John Abelson, for a mini-sabbatical, to learn the ‘plus-minus’ DNA sequencing method, which used these labelled triphosphates for in vitro synthesis of short products.

I have enjoyed sharing stories and experiences with those who were in Fred’s lab either before or after I was. Beverly Griffin, who preceded me and then went to ICRF, used to tell me how masterful Fred was in leading on someone who might be a little shaky on all the facts under discussion. She was right, as I found out on a few occasions.

FRED’S GROUP

Like most other group leaders at the MRC, Fred did not have many postdocs or students. When I arrived George Brownlee had just received his degree, sequencing 5S ribosomal RNA, and Peter Fellner was still a research student. Fred had one other postdoc in his lab, Shyam Dube, who was collaborating with Kjeld Marcker on studies on tRNAf-met. Shyam was always good for a laugh and a fun story, to pick up my spirits after a sheet of wet DEAE paper had fallen apart and collapsed onto the bench (or the floor). I shared space with George, Peter and Bart Barrell for about a year, before moving down the hallway to the room where Kjeld and Ken Murray worked.

Ken (later to become Sir Kenneth Murray) was working on methods to sequence DNA. In those days we were all focused on digestion and analysis of nucleic acid fragments (RNA or DNA) but sequence-specific restriction enzymes were not yet in play. Ken was also developing a spark chamber that could amplify and quantify signals from the decay of 32P. As everyone worked with millicurie levels of 32P on a regular basis, we were eager to devise methods that would reduce the amounts needed in our experiments. Unfortunately, spills were not uncommon, leading another postdoc, Art Landy, to remark: ‘At the MRC one washes his hands before, rather than after, going to the bathroom.’

THE CANTEEN

Everyone who has been through the MRC lab has endless stories to tell about the canteen, regarding Mrs Perutz, the food, and the discussions held at morning coffee, lunch and afternoon tea. In large part, the exciting and stimulating environment throughout the laboratory grew out of the atmosphere there, which was an ever-fertile font of ideas and discussion. The canteen was not just a place in which to eat or have coffee or tea, but a phenomenon with its own purpose. I have tried to instill this concept into any laboratory or department I have been associated with: regardless of rank, everyone is to be considered with equal dignity and respect for ideas, and open discussion is strongly encouraged. Very early in my stay at the laboratory I was first exposed to this openness in conversations with Gobind Khorana, who was on sabbatical from the University of Wisconsin (and who later was influential in helping me get a job there).

The canteen was a hub for cross-fertilisation of ideas that came from many disciplines: protein and nucleic acid sequencing, molecular genetics and structural biology, all mixed with lots of free-association and critical thinking. Most of the group leaders went regularly to these sessions, but Fred seemed not to do so very much; I think he did not like spending so much time away from his bench. Interactions there that were memorable for me include a discussion with Francis Crick about why complexes such as messenger RNAs bound to ribosomes can survive sedimentation in sucrose gradients without falling apart; because all complexes transiently dissociate, Francis argued that the complexes should not survive, as constituents would be separated from each other during the spin. I think that he knew the answer and was just enjoying a good discussion. I also enjoyed talking with César Milstein and Blas Frangione about their sequencing of antibodies that were purified from patients with multiple myeloma; this was before César discovered how to make monoclonal antibodies.

Several stimulating hours were spent discussing with Aaron Klug an idea I had about isolating specific oligonucleotides from complex mixtures. The idea arose while talking with Brian Hartley, who had devised a two-dimensional paper electrophoretic ‘diagonal’ method to identify oligopeptides in digests of enzymes such as elastase. These discussions led me to devise a diagonal method for isolation of ends of RNAs; Harry Noller, who was in Ieuan Harris’s lab at the time, later used similar methods to study kethoxal-accessible regions in 16S ribosomal RNA, in his own lab at UCSC. My RNA diagonal work allowed me to isolate and sequence the ten nucleotide-long RNase T1 oligonucleotide from the 3′ end of phage f2 RNA, resulting in a single-author paper in Nature (my only one). It was a sign of the times, that a sequence of ten nucleotides could suffice for that!

MULTI-TASKING

I have always found it stimulating to work on several projects in parallel. Initially, in Fred’s lab I was interested in following up on my PhD thesis project, the characterisation of ribosome binding sites in messenger RNAs. For that I had started working with phage f2, with which Harvey Lodish (working in Sydney’s lab) furnished me. I later abandoned this project when Joan Steitz expressed great interest in working on the ribosome binding sites, and I was plenty busy with other experiments; Joan obviously did very well with it. In addition to the isolation of the ends of RNAs, and initial work on ribosome binding sites in f2 RNA, I also collaborated with Brian Clark, Mark Bretscher and John Anderson, characterising some translation initiation factors.

I recall discussing this sort of experimental multi-tasking with Aaron at tea one day. I lamented that if I am doing several things successfully, but one experiment does not work as hoped, I dwell on the unsuccessful one, not on the successful ones. I was gratified to hear that Aaron had the same reaction.

MANY EXCITING DISCUSSIONS

I regret not spending more time on Saturday mornings in the second-floor kitchen where Sydney would hold open-ended discussions with postdocs from all areas of the laboratory. Sydney was very interested in developing a new eukaryotic model organism and was particularly taken by the advantages of C. elegans. Many of the postdocs were eager to participate in this endeavour, but at that time (1967–8) Sydney felt that it was premature for a postdoc to risk several years on something that might not work out; soon afterwards, of course, this system became a major focus of his lab.

Another exciting topic of conversation around the lab centered on the determination of the sequence of the Su3+ suppressor tRNAtyr being determined in John Smith’s lab, by Howard Goodman, John Abelson and others. The discovery of the mutation in the anticodon confirmed everyone’s expectations and was followed by investigation of the nature of the modification of the nucleotide just 3′ of that region. A big surprise that came soon afterwards was David Hirsh’s discovery of a change in the D-loop of a tRNAtrp affected suppression of a UGA codon.

OTHER THAN SCIENCE

Of course, most of my conversations in the canteen were with fellow postdocs and a few graduate students. Although we talked a lot about our experiments and ideas, we also discussed much more than science. The first year I was in Cambridge (1966), the big topic of conversation was the celebration of the 900th anniversary of the Battle of Hastings, plus a big rugby game at Wembley between two teams whose names I cannot recall.

The group of postdocs and students with whom I interacted on a daily basis included John Abelson, Harry Noller, Tom and Joan Steitz, Peter Moore, Sid Altman, Malcolm Gefter, Art Landy, David DeRosier, Gary Borisy, Harvey Lodish, Suzanne Cory, Jerry Adams, Andrew Travers, George Brownlee, Steve Martin (who did amazing impersonations) and Bart Barrell (who at the time was not yet a research student). Quite a crew, when I look back on it!

I connected with John Abelson even before going to Cambridge when Lucy (my wife at the time) and I drove to Greece in the summer of 1966 to attend a Spetsai workshop/summer-school. John was also attending this course, accompanied by his wife and daughter (Lynne and Sara). We all hit it off right away, and I even recall John defending me in an incipient scuffle in a Greek bar.

OUTSIDE THE LABORATORY

I have to admit to being quite naïve when I first came to Cambridge. Because of our excitement in being in Cambridge, Lucy and I thought that nothing could be unpleasant anywhere in the entire city. Thus we rented a flat on Tenison Road, near the train station for ourselves and our newly acquired springer spaniel, Emma. We were a bit disappointed when we proudly showed our newly rented flat to John and Lynne Abelson, who were less than enthusiastic about the row house that we had chosen. I realised that John and Lynne were right and that we were in big trouble when we woke up freezing in the middle of August. Fortunately we were able to get out of our lease and move out to a nice, but mundane, house in Stapleford, south of Cambridge, near Great Shelford.

Another naïve trait was to think that pounds and dollars were equivalent, so one of my first purchases was a very nice sheepskin coat that cost about twice what we could afford. But we recovered and were even able to save enough money to purchase some beautiful Georgian antiques from an antique dealer in Bury St Edmunds, Mrs Barbara Usher, whom we had befriended.

Much of our social life revolved around activities with other postdocs, both in Cambridge and on day trips to London. The conversation at dinner parties in our homes often turned to what was, at the time, a big controversy: the impending publication by Jim Watson of The Double Helix, his account of the discovery of the structure of DNA. Francis was apparently trying to block its publication (presumably because of the way he was portrayed).

For fancy meals several of us would go out to dinner at the Pink Geranium in Melbourn. We also made day trips to London, taking advantage of the fact that a one-day return trip cost only 21 shillings (about $3) on British Rail. Some shopping, dinner and a show made for a great excursion. For eating out in London we relied on a book called The London Good Food Guide, which David DeRosier (a fellow ex-graduate student from Chicago, working with Aaron) pointed out cost less than a terrible meal in London. I recall going to wonderful concerts in London, as well as seeing the play The Prime of Miss Jean Brodie, starring Vanessa Redgrave, and a disturbing play about the US involvement in Vietnam, called US. Lucy and I also spent time socially with Ken Stevenson and his wife Barb, from Edmonton, Alberta; I believe that Ken was a postdoc with Brian Hartley.

OTHER ACTIVITIES

Another extracurricular activity that I briefly considered was to become part of the initial crew of scientist-astronauts in NASA. In 1967 or 1968 NASA was interested in actually doing some science in space and they contacted several individuals, inviting them to apply. I went so far as to send a telegram to NASA asking for information and an application form. I phoned the message into the telegram office, and from the operator’s reaction I got the feeling that she thought John Glenn must have been on the line. In the end, several factors led me not to apply: not knowing what I would do after I was too old to be an astronaut, and most importantly, the fact that if I became part of that corps, I would have to live in Houston. However, Howard Goodman did apply and my understanding is that he made it through several levels of screening before being eliminated.

One of the highlights of my musical experience in Cambridge was when Fred gave Lucy and me tickets to sit in the choir seats at King’s during the 1967 Christmas Festival of Nine Lessons and Carols, broadcast live around the world by the BBC. Fred was not much into music and he knew that we were, Lucy being a serious amateur harpsichordist: she took lessons from Mary Potts, whose house and studio were, I believe, on Bateman Street, near Hills Road. We had purchased a small harpsichord from the owner of the Cambridge Music Shop in the centre of town (just off Sidney Street). Another musical highlight, among many, was listening to the Brahms Requiem in King’s Chapel.

AFTER CAMBRIDGE

Harry Noller, Howard Goodman, Peter Moore, Jerry Adams, Suzanne Cory and I supplemented our postdoctoral training in Cambridge by spending time at the Institut de Biologie Moléculaire, in Geneva. I continue to interact with most of these people, plus John Abelson and Joan and Tom Steitz, both at meetings and on joint vacations. These friends, along with many of the other American postdocs at the time, are now members of the US National Academy of Sciences (attesting to the enduring influence of the MRC), so we often meet at the annual meeting of the NAS.

Today, I count several of my colleagues whom I met at the MRC as some of my closest friends; I still take annual skiing vacations with John, Joan and Tom, and enjoy a week of trout fishing with Harry, every summer. Also, I have spent many happy days fishing for salmon off the west coast of Canada, with John, Harry and Peter.

LOOKING BACK

While I was in Cambridge, several members of my family came to visit. I recall telling my brother that I realised these would turn out to be some of the best and happiest years of my life. I was right. There is a certain feeling of shared experience that I have when associating with other former postdocs from the MRC-LMB, even if we did not overlap in time. We all have been exposed to the same exciting thought processes and thrill of discovery in that very special place, during the golden age of molecular biology.

In addition to establishing lifelong friends and colleagues, my experiences at the laboratory affected my scientific career. After leaving Cambridge, when I was a postdoc in Switzerland I collaborated with Howard Goodman, John Hindley, Martin Billeter and Charles Weissmann on the sequencing of 175 nucleotides at the 5′ end of the RNA of phage Qβ, which we synthesised in vitro. After arriving at the University of Wisconsin I applied the same methodologies to sequence mRNAs of phage λ and also used RNA fingerprinting to identify tRNAs that serve as primers for retrovirus reverse transcription. Later, with Elsebet Lund, my wife and long-time collaborator, I investigated synthesis and processing of ribosomal RNAs, tRNAs and small nuclear RNAs. Although neither of us worked with John Gurdon in Cambridge, in Madison we have used Xenopus laevis oocytes extensively, as a powerful and convenient system in which to study transport and maturation of eukaryotic rRNAs, tRNAs, snRNAs and microRNAs.

Looking at my career, I realise that Fred taught me the importance of staying in the lab and keeping my research group small; unfortunately, with the passage of time I found it more and more difficult to stay out of my office. Also, from Fred I learned how the development of new methodologies makes it possible to ask new questions; from Sydney I learned to choose important but manageable problems; from Aaron I learned the power of symmetry in biology; from Francis I learned the importance of seeing the essence of problems, not being distracted by irrelevant details. All of these lessons have served me well, and I have always tried to ask questions that can yield unambiguous answers. Probably the most important question that I often ask is: ‘What would Fred do here?’

CHAPTER 18

Meeting at the Mecca: the LMB in the late 1960s

Suzanne Cory and Jerry Adams

Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

Figure 18.1. Joan Steitz, Tom Steitz, Jerry Adams and Suzanne Cory in Switzerland, c.1968. Photo by Mark Bretscher.

Australian Suzanne Cory and American Jerry Adams, who met at the LMB in 1966, describe why they came here, what the lab was then like and its impact on their subsequent successful joint careers in Australia.

SUZANNE

DNA first began to fascinate me during an introductory genetics unit at the University of Melbourne when I heard that each of our chromosomes contained a single giant molecule of DNA – the concept of a single molecule encoding thousands of genes blew my mind. The next year, in biochemistry, I learned hot off the press about the cracking of the genetic code and my fascination leaped again. At the end of my undergraduate course I was invited to do an MSc in the biochemistry department and, guided by Lloyd Finch, I gained skills in isolating and fractionating RNA. I found that I enjoyed lab life and, after the heady experience of my first scientific conference (the annual meeting of the Australian Biochemistry Society), I decided to undertake a PhD. But not in Melbourne. With the naïve audacity of youth, which I look back upon with amazement, I sought to go to the Mecca of molecular biology, the LMB, and wrote to Francis Crick asking if I could do my PhD in his lab. I am still surprised he took me on, although I later learned that the head of my department, Victor Trikojus, had visited the LMB to plead my case.

After six wonderful months’ youth hostelling around Europe, I arrived at Cambridge in September 1966. I had been very fortunate to win a scholarship from the Royal Commission for the Exhibition of 1851 – fortunate because there were then very few Australian scholarships to study overseas, and I found all were explicitly restricted to men! I became the first student of a new postgraduate college, University College (now Wolfson), which I had learned on Australian radio was open to both women and men. I lived in two small whitewashed attic rooms in Bredon House in Selwyn Gardens, with a wonderful view of the extensive gardens. It was very different from the traditional Cambridge colleges, which were all still segregated, but its informal, friendly atmosphere suited me.

By then, of course, I had learned all about the LMB with its ‘Nobel laureates on every floor’. I still vividly remember the first day, walking up the stairs past the giant model of tobacco mosaic virus and the photo of Lawrence Bragg, to front up to the daunting experience of being interviewed jointly by Francis Crick and Sydney Brenner. They told me they had decided I should work on the sequence of a methionine tRNA with Brian Clark as my supervisor. Sydney growled, ‘Do you think you are up to it?’; I stammered ‘yes’ (although privately I was not so sure) and was given a small corner of bench in Mark Bretscher’s laboratory in their molecular genetics division.

Brian Clark had joined the LMB two years earlier, after a postdoctoral period with Marshall Nirenberg at NIH, where he had become skilled in cell-free protein synthesis. Kjeld Marcker and Fred Sanger had recently discovered N-formyl methionyl-tRNA1 and experiments from Cambridge2 and Harvard3 (see below) had revealed this to be the polypeptide chain initiator in bacteria. Bretscher, Clark and Marcker4,5 found that, unlike any other loaded tRNAs, formyl-methionyl-tRNA was sensitive to puromycin, an analogue of amino acyl-tRNA, leading them to postulate that it bound only to the ‘P site’ on the ribosome. The other methionyl-tRNA inserted Met into internal positions in polypeptides, presumably because it could only bind to the ‘A site’. In June 1966, Clark and Marcker had decided that the complete nucleotide sequence of both Met tRNAs should be determined, to clarify why they had such different properties. Shyam Dube, a US postdoc with Marcker, and I were to take on the challenge.

When we started, it was not clear which strategy to take. Holley had just completed the first tRNA sequence6 using conventional biochemical procedures, but this had required copious quantities of pure tRNA and had taken a large team several years to complete. In contrast, Sanger’s lab had just developed innovative methods for sequencing microgram quantities of 32P-labelled RNA, which they had successfully applied to 5S ribosomal RNA,7 and promising results were being obtained with a genetically amplified tyrosine suppressor tRNA.8 We strongly favoured the Sanger procedure, but it was unclear whether the less abundant Met-tRNAs could be purified quickly enough, given the short half-life of 32P (fourteen days). It was therefore decided (before I arrived) that Shyam and Kjeld would try the radioactive approach, while Brian and I would undertake large-scale purification of nonradioactive Met tRNAs.

This approach was real bucket biochemistry. Due to the immense scale required – 1,000 litres of bacterial culture to obtain 100g of crude tRNA – Brian had persuaded the Microbiological Research Establishment at Porton to grow the E. coli cells and carry out the initial phenol extraction of crude tRNA. The plan was then to fractionate the 60 or so different tRNA species using countercurrent distribution (CCD), which depends on differential distribution between two immiscible solvents. Brian introduced me to his three daunting CCD machines with their large banks of stoppered tubes and I set to. I eventually succeeded in isolating pure Met tRNAM, the first to do so.9 By then, however, Dube and Marcker had purified 32P-labelled MetF tRNA and were sequencing its component oligonucleotides, and I persuaded Brian to let me try the faster and more elegant radioactive approach for Met tRNAM. He persisted with the large-scale purification work to enable 3D-structural studies.10

I devised a fractionation procedure that enabled me to rapidly purify both 32P-labelled Met tRNAs, allowing Shyam to complete the MetF tRNA sequence11 and me to sequence MetM tRNA.12 The ‘fingerprinting’ sequencing procedure7 was a real craft and I was fortunate to be taught by masters – in particular Shyam, George Brownlee and Bart Barrell. The RNA was cleaved with a base-specific ribonuclease (e.g. T1 RNase, which cleaves after G residues) and the oligonucleotide mixture resolved by very high-voltage 2D paper electrophoresis in large tanks of coolant. The resulting ‘fingerprint’ was revealed by autoradiography. Clues to the sequence of each oligonucleotide spot came from the RNase specificity, the position of the spot on the ‘graticule’ and its base composition. The order of the primary products within the tRNA sequence was deduced by analysing larger fragments produced by partial digestion. Basically, it was like doing a jigsaw puzzle. I can still remember precisely where I was walking in Cambridge when the last piece of the jigsaw puzzle dropped into place in my mind and I knew my PhD was complete!

Looking back, these were heroic times. Today, no PhD student would ever be permitted to label bacterial cells starting with 100 milliCurie of 32P. We worked behind thick plastic shields, but even a minute spill would produce terrifying screams from the Geiger counter!

Impressions of the LMB

Life at the LMB made an indelible impression and taught me many things. Its scientists worked with a dedication and determination to succeed that I had barely glimpsed before. They were in a race with the rest of the world to unlock the secrets of life and they intended to be first! The daily excitement was palpable. These lessons became imprinted on my consciousness. I saw that science was a vocation, not a job, and was only worth doing if you asked the big questions and were fully committed. The diversity of the memorable personalities around me, ranging from the incisive but domineering Francis Crick and Sydney Brenner, to the modest but equally influential Fred Sanger and Max Perutz, taught me that genius has many guises. I also learned the importance of access to the latest technologies and equipment and how these can transform a field. I saw that success breeds success – highly talented postdocs were attracted to the superstars at the LMB like bees to a honeypot, and their ambition to succeed helped drive the next major discoveries and opened up their own career opportunities.

Most important to me personally, it was through the LMB that I met Jerry Adams, who was to become my husband and lifelong scientific partner. I actually knew about Jerry before I met him, because the very first scientific talk I ever gave, at a scientific journal club in Melbourne, was on the paper he had just published with Mario Capecchi on FMet-tRNA. I had been amazed at the time that the authors of this exciting paper were still PhD students! So I was most intrigued when I learned that Jerry was coming from Jim Watson’s lab to do a postdoc with Fred Sanger. It was Max Perutz who introduced us, just after Jerry’s arrival, at the Christmas party he hosted for the LMB every year at his college, Peterhouse. Somehow Max had learned that Jerry and I were both ski enthusiasts and thought these two newcomers might like to meet. Jerry almost blew his opportunity, however, by complimenting me on my nice ‘British’ accent!

During the long hours spent in the lab, I got to know many remarkable staff scientists, postdocs and students, from whom I learned a great deal. Mark Bretscher’s lab was a fun place. Mark himself was an excellent source of scientific advice – and scientific gossip. Susan Brownlee, his research assistant and George Brownlee’s wife, was a keen musician and helped me join the acclaimed choir of the Cambridge University Musical Society, then directed by the inspiring David Wilcox. In 1967, Joan Argetsinger Steitz joined Mark as a postdoc and embarked upon studying the ribosome binding sites of the R17 phage genome. Joan and her husband Tom Steitz, an X-ray crystallographer working on chymotrypsin in David Blow’s lab, had been fellow PhD classmates of Jerry at Harvard. They were to play a crucial role in encouraging our relationship, which had its ups and downs in the early days – I was keen to return to Australia and entanglement with an American seemed likely to thwart this goal.

Sydney Brenner had already embarked on his epic mutagenesis of C. elegans and was always tightly secluded, either in his lab or in the office he shared with Francis on the first floor. But every Saturday morning he held court over coffee, sitting on the bench in the media kitchen on the second floor, regaling us with ‘behind the scenes’ accounts of the earliest days of molecular biology and the LMB. If only I could remember even a fraction of those stories! Often present were Harvey Lodish, Art Landy, John Abelson and Paul Sigler, all talented postdocs from the US, and Steve Martin, Sydney’s PhD student, who spoke just like Sydney; whether from irony or admiration I could not tell.

Because the electrophoresis equipment and chromatography tanks were upstairs on the third floor, I also got to know many of the protein chemists, including César Milstein, while chatting around the huge glass-topped table used to wet up paper for electrophoresis. It was there that I also met Ken Murray and Moshe Yaniv, who came for short visits to learn nucleotide sequencing from Fred. Like Tom and Joan Steitz and the Brownlees, they became our lifelong friends, as did Jim Dahlberg and Howard Goodman, nucleic acid sequencers who preceded us at LMB but with whom we overlapped later, in Geneva.

The epic discovery of the structure of DNA once again became highly controversial during this period, as drafts of Jim Watson’s remarkable personal account The Double Helix circulated between Harvard and the LMB. As Bragg wrote in the foreword, Jim wrote with ‘Pepys-like’ frankness, and his candour created a bitter (albeit temporary) rift between Francis and Jim. Watson’s patronising portrait of Rosalind Franklin rankled deeply with me, and I was very pleased when Aaron Klug defended her major contribution in Nature after Jim’s book was published (1968).

Even though I lacked sufficient training in physics to ever master X-ray crystallography, its all-pervasive influence at the LMB made an indelible mark and I was later to recruit a structural biology division to our institute in Melbourne, which greatly enriched our capability.

The tapestry of my memories of life in Cambridge has many colours and patterns: the soaring music in King’s College; biking along snow-edged paths in the winter; summers with strawberries and double cream, champagne, punting and May Balls; Sunday roasts in picturesque pubs (I could never get used to warm beer but the ciders were wonderful); dancing rock and twist; short short skirts, Kashmiri sheepskin jackets and flamboyant ties; and visits to ‘swinging London’ to shop in Carnaby Street and Chelsea Road and experience great opera and theatre.

JERRY

Excited by the new discipline of molecular biology, I came from Emory University in Atlanta, Georgia to Harvard in 1962 to do a PhD and was very fortunate that Jim Watson took me on as a student. It was a heady time to be entering this new science, particularly in that remarkable lab. The DNA double helix was only a decade old and its many ramifications were still being uncovered. Bacterial messenger RNA had only recently been discovered (partly in the Watson lab); Wally Gilbert, who shared the lab with Jim, was avidly hunting the lac repressor; Crick’s adaptor hypothesis for transfer RNAs was still young; and the genetic code – the Rosetta Stone of biology – was beginning to be deciphered.

As a thesis project, Jim set me to clarify the initiation of bacterial polypeptide chains. Kjeld Marcker and Fred Sanger at the LMB1 had recently provided an important clue by showing that some of the methionine attached to a transfer RNA had its amino group blocked by a formyl residue. If the formyl-methionine contributed to protein synthesis, it could only be inserted as the first residue in the chain, revealing a special mechanism for polypeptide initiation. I devised a way to label the formyl group on methionine tRNA and then collaborated with fellow PhD student Mario Capecchi – decades later to receive the Nobel Prize (2007) for developing the gene knockout technology – to show that indeed formyl-methionine could be incorporated into protein, even for a polypeptide whose mature form lacked an N-terminal methionine.3 In other words, formyl-methionine tRNA was a universal polypeptide initiator, a major finding. Jim then suggested that I follow up by characterising the enzyme that removed the formyl group from nascent polypeptides. I devised a good assay using formyl-peptides and characterised the activity, but the deformylase proved so labile that four frustrating years of effort yielded no purification!13 It is fortunate that I eventually gave up, because its profound instability was only overcome three decades later!

For my postdoctoral studies, I chose the LMB because it was the symbolic birthplace of molecular biology and the world’s most influential centre in the field. I had seen that illustrated at the exciting 1966 Cold Spring Harbor Symposium on the Genetic Code, which was dominated by Francis Crick, who cajoled all those working on the code to reveal their unpublished findings, so that a definitive version finally emerged as the frontispiece of the volume. I asked Fred Sanger to take me on because I surmised that a deep understanding of biology would require knowing the precise structure of many of the nucleic acids, and his unique techniques were the most promising. And of course there was the strong appeal of going abroad for the first time and seeing more of the world.

The Sanger sequencing technique had been successfully applied to small RNA molecules such as transfer RNAs (70–90 n) and the 5S ribosomal RNA (120 n), but when I arrived the lab had begun to try sequencing large RNA molecules, using R17 phage RNA (~3000 n) as a model messenger. A new PhD student with Fred, Peter Jeppesen, had been able to isolate and sequence several large oligonucleotides from complete T1 RNase digests of R17 RNA, but it was unclear how larger sequences could be obtained. I thought that the RNA might have enough structure to allow fragmentation by partial digestion. The test was facilitated by electrophoresis of the digests on flat polyacrylamide gels (between glass plates), a technique that George Brownlee had just devised. To my delight, the partial digests yielded a number of R17 RNA fragments pure enough for sequencing. Fortunately, one of the sequences proved to encode a known amino acid sequence from the R17 coat protein. Hence, the study provided the first direct evidence for the validity of the recently elucidated genetic code.14

Fred Sanger and Jim Watson, undoubtedly among the very greatest scientists of the 20th century and both wonderful mentors, represented opposite approaches to science. To Jim, concepts were always paramount, and techniques were what you picked up to do the critical experiment testing an idea. Fred, on the other hand, was the supreme experimentalist, never happier than when he was using his own hands. His ultimate goal was to devise new universal methods. He had amply demonstrated the importance of new methodology in determining the first sequence of a protein (insulin), for which he received his first Nobel Prize (in 1958), and in devising the best approach to RNA sequencing; and later he developed the DNA sequencing method that not only led to his second Nobel Prize (1980) but laid the foundation for the Human Genome Project.

Despite Fred’s immense achievements, he remained a most modest and gentle man. Once, in discussion with him, I put my foot firmly in my mouth by derisively describing a publication to him as ‘just a methods paper’. Fred gently remonstrated: ‘I should have thought that a methods paper can sometimes be helpful.’ Indeed!

Personally, my biggest challenge at the LMB was convincing a bright and very attractive but strong-willed young Australian woman to cast her fortunes in with mine. Fortunately for me, persistence won out, and our scientific and personal lives have now been happily entwined for over four decades.

JERRY AND SUZANNE: LIFE AFTER LMB

Geneva

Jerry had already organised to spend a period after Cambridge at the Institut de Biologie Moléculaire headed by Alfred Tissières at the University of Geneva. We arrived in March 1969, after our marriage in Melbourne. Since securing an apartment was always a challenge in Geneva, new postdocs tended to ‘inherit’ the abodes of those departing. Thus we spent our first few months in a ‘menage a trois’ with Jim Dahlberg, before he took up a position in Madison. Jim and Howard Goodman, as well as introducing us to the delights of living in Geneva – wine, food, skiing and hiking – had ensured that the lab was well equipped for Sanger RNA sequencing.

We continued the work Jerry had started at Cambridge, using R17 phage RNA as a model for messenger RNA. We discovered unexpected secondary structure and untranslated regions,15–18 and Suzanne presented the findings at the 1970 Cold Spring Harbor Symposium. R17 sequencing was also continuing at the LMB, and we were honoured to have Fred Sanger stay with us when he visited Geneva for discussions.

The remarkable gymnastics of antibody genes

While in Geneva, we decided to move to Australia, and successfully applied to the Walter and Eliza Hall Institute, the immunology institute in Melbourne brought to world fame by Nobel laureate Macfarlane Burnet and now brilliantly led by Gus Nossal. We judged the time was ripe to move from prokaryotes to eukaryotes and hoped to use molecular biology to solve the major questions then being asked by immunologists, most notably how the body generates the billions of different antibodies it uses to fight off disease.

We arrived in late 1971, set up a high voltage electrophoresis lab modelled on that at the LMB, and began to isolate 32P-labelled immunoglobulin mRNA from mouse plasmacytoma cell lines. Unfortunately, it proved impossible to purify enough of the specific mRNA for sequencing studies, but we did make a major discovery about mammalian messenger RNA. Fingerprints yielded unexpected oligonucleotides that we eventually deduced to be 7-methyl-guanosine in a novel 5′, 5′ triphosphate linkage on the 5′ end of the messengers.19 Fred Sanger happened to visit just as we were preparing the paper. We laid out our evidence for these bizarre mRNA ‘cap’ structures and anxiously awaited his reaction. Fred stood quietly for a moment and then delivered his typically understated ultimate accolade, a smiling ‘That looks about right!’

Our original objective only became feasible in the mid-70s with the recombinant DNA revolution. With strong support from Nossal, we raced to set up the elaborate CII containment facilities then required because of prevailing global fears of creating an ‘Andromeda strain’, and used our partially purified messenger RNAs to clone immunoglobulin (Ig) cDNAs and their genes,20,21 aided by a workshop on lambda phage cloning run in Australia by Ken and Noreen Murray in 1976. The surprising answer to the puzzle of antibody diversity that emerged from several laboratories, including that of Terry Rabbitts at the LMB, was that the N-terminal regions of antibodies are encoded in the germline as clusters of V (variable), D (diversity) and J (joining) elements, and that each lymphocyte must actively assemble a complete Ig gene from these components, selected at random. The immense combinatorial possibilities permit the generation of enormous numbers of antibody specificities. Our major contribution during this exciting period was to show that these genetic recombination events in lymphocytes deleted the intervening DNA.22

Cancer genetics

In 1975, Jerry and I had been electrified to learn that Harold Varmus and Michael Bishop had found that every mammalian cell contains genes resembling those in acute leukaemia viruses from birds, rodents and cats. The implication was that our genome harbours the seeds of cancer, genes that can turn rogue if mutated or expressed in the wrong cell type, or at the wrong time. Then, in 1981, Hayward, Neel and Astrin found that avian leukaemia could result from activation of a cellular oncogene by the nearby insertion of a retrovirus that itself contained no oncogene. We began reading voraciously in the new field, keen to join the ‘oncogene’ hunt. Pertinently, cytogeneticists had just published that the chromosome translocations that hallmark mouse plasmacytomas and human Burkitt’s lymphomas (both tumours of antibody-producing cells) involved chromosomes harbouring Ig gene loci. Knowing that antibody gene assembly involves intra-chromosomal cutting and re-ligation, we hypothesised that the translocations were aberrant antibody gene rearrangements that had coupled an antibody gene locus to an oncogene on the partner chromosome.

To test this hypothesis, we used an antibody gene as a tag to clone the putative linked oncogene. Indeed, each tumour proved to contain an antibody gene linked to the gene myc, the mammalian counterpart of a known avian oncogene,23 a finding also made independently by others. While myc seemed highly likely to be the culprit, these findings only provided guilt by association. To seek direct proof, we turned to transgenesis, a new technology then being pioneered by Richard Palmiter and Ralph Brinster. We sent them a cloned inter-chromosomal junction from a plasmacytoma that contained myc linked to an Ig gene enhancer. They injected the DNA into mouse oocytes and monitored the transgenic offspring. Remarkably, every mouse succumbed to a disseminated lymphoma within twelve months, proving that activation of myc by chromosome translocation was the root cause of Burkitt’s lymphoma and mouse plasmacytoma.24 Our lymphoma-prone myc mice became a powerful model that is still used around the world for studying tumour development.

The genetic control of cell survival

The myc translocation became a paradigm for isolating many other oncogenes, from different kinds of lymphomas and leukaemias, and our lab used transgenesis to study the physiological impact of several. The most illuminating was bcl-2, which had been cloned (by others) in 1984 from the chromosome translocation associated with follicular lymphoma. Its function remained a mystery until 1988, when David Vaux discovered during PhD studies in our lab that over-expression of Bcl-2 promotes cell survival under adverse growth conditions.25 With David and Andreas Strasser, we went on to show that over-expression of Bcl-2 is indeed oncogenic, especially when combined with activation of myc.25,26 These studies revealed for the first time that cancer development involves not only genetic accidents that promote cell proliferation, but also those that block apoptosis, the normal cell death process.

Bcl-2 proved to have a score of relatives, and while some of them also promote cell survival, others instead promote cell death. Together, they constitute a critical switch that determines whether a cell lives or dies in response to the complex signals it receives internally and from its environment. The Bcl-2 family has become our central focus.27 Several labs within our Institute now work together to decipher the molecular circuitry of cell death and use this knowledge to help develop more effective drugs and regimens for eliminating cancer cells.

The long-term impact of our LMB experiences

At first glance, our scientific journey over recent decades may not seem closely linked to our experience at the LMB. However, all our work has involved the molecular genetics of important biological phenomena. Furthermore, the seminars we heard from César Milstein’s group about antibody sequences and their diversity greatly influenced our decision to move into immunogenetics in Melbourne and that in turn led inexorably to cancer genetics. More generally, the attitudes that we acquired at the LMB have remained with us always: asking the big questions, embracing emerging technologies and developing long-term relationships with colleagues having similar aims and commitment. We greatly value the many friendships we made at the LMB and remember all those who influenced us there, particularly Fred Sanger, with great respect and affection.

Suzanne Cory and Jerry Adams were elected Fellows of the Australian Academy of Science in 1986 and Fellows of the Royal Society in 1992. From 1996 to 2009, Suzanne served as Director of the Walter and Eliza Hall Institute. In 2010, she was elected for a four-year term as President of the Australian Academy of Science. Suzanne was elected a Foreign Associate of the US National Academy of Sciences in 1997, and Jerry was elected a Member of that Academy in 2008.

REFERENCES

1. K. Marcker, F. Sanger, 1964. N-Formyl-Methionyl-S-RNA. Journal of Molecular Biology 8, 835–40.

2. F.C. Clark, K.A. Marcker, 1965. Coding response of N-formyl-methionyl-sRNA to UUG. Nature 207, 1038–9.

3. J.M. Adams, M.R. Capecchi, 1966. N-formylmethionyl-sRNA as the initiator of protein synthesis. Proceedings of the National Academy of Science of the United States of America 55, 147–55.

4. B.F. Clark, K.A. Marcker, 1966. The role of N-formyl-methionyl-sRNA in protein biosynthesis. Journal of Molecular Biology 17, 394–406.

5. M.S. Bretscher, K.A. Marcker, 1966. Polypeptidyl-sigma-ribonucleic acid and amino-acyl-sigma-ribonucleic acid binding sites on ribosomes. Nature 211, 380–84.

6. R.W. Holley, J. Apgar, G.A. Everett et al, 1965. Structure of a Ribonucleic Acid. Science 147, 462–5.

7. F. Sanger, G.G. Brownlee, B.G. Barrell, 1965. A two-dimensional fractionation procedure for radioactive nucleotides. Journal of Molecular Biology 13, 373–98.

8. H.M. Goodman, J. Abelson, A. Landy, S. Brenner, J.D. Smith, 1968. Amber suppression: a nucleotide change in the anticodon of a tyrosine transfer RNA. Nature 217, 1019–24.

9. B.P. Doctor, B.J. Wayman, S. Cory, P.S. Rudland, B.F. Clark, 1969. Studies on the Escherichia coli Methionine transfer ribonucleic acids. European Journal of Biochemistry 8, 93–100.

10. B.F. Clark, 2001. The crystallization and structural determination of tRNA. Trends in Biochemical Sciences 26, 511–14.

11. S.K. Dube, K.A. Marcker, B.F. Clark, S. Cory, 1968. Nucleotide sequence of N-formyl-methionyl-transfer RNA. Nature 218, 232–3.

12. S. Cory, K.A. Marcker, S.K. Dube, B.F. Clark, 1968. Primary structure of a methionine transfer RNA from Escherichia coli. Nature 220, 1039–40.

13. J.M. Adams, 1968. On the release of the formyl group from nascent protein. Journal of Molecular Biology 33, 571–89.

14. J.M. Adams, P.G. Jeppesen, F. Sanger, B.G. Barrell, 1969. Nucleotide sequence from the coat protein cistron of R17 bacteriophage RNA. Nature 223, 1009–14.

15. J.M. Adams, S. Cory, 1970. Untranslated nucleotide sequence at the 5′-end of R17 bacteriophage RNA. Nature 227, 570–574.

16. J.M. Adams, S. Cory, P.F. Spahr, 1972. Nucleotide sequences of fragments of R17 bacteriophage RNA from the region immediately preceding the coat-protein cistron. European Journal of Biochemistry 29, 469–79.

17. J.M. Adams, P.F. Spahr, S. Cory, 1972. Nucleotide sequence from the 5′ end to the first cistron of R17 bacteriophage ribonucleic acid. Biochemistry 11, 976–88.

18. S. Cory, J.M. Adams, P.F. Spahr, U. Rensing, 1972. Sequence of 51 nucleotides at the 3′-end of R17 bacteriophage RNA. Journal of Molecular Biology 63, 41–56.

19. J.M. Adams, S. Cory, 1975. Modified nucleosides and bizarre 5′-termini in mouse myeloma mRNA. Nature 255, 28–33.

20. D.J. Kemp, S. Cory, J.M. Adams, 1979. Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome. Proceedings of the National Academy of Science of the United States of America 76, 4627–31.

21. J.M. Adams, D.J. Kemp, O. Bernard et al, 1981. Organization and expression of murine immunoglobulin genes. Immunological Reviews 59, 5–32.

22. S. Cory, J.M. Adams, 1980. Deletions are associated with somatic rearrangement of immunoglobulin heavy chain genes. Cell 19, 37–51.

23. J.M. Adams, S. Gerondakis, E. Webb, L.M. Corcoran, S. Cory, 1983. Cellular myc oncogene is altered by chromosome translocation to an immunoglobulin locus in murine plasmacytomas and is rearranged similarly in human Burkitt lymphomas. Proceedings of the National Academy of Science of the United States of America 80, 1982–6.

24. J.M. Adams, A.W. Harris, C.A. Pinkert et al, 1985. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318, 533–8.

25. D.L. Vaux, S. Cory, J.M. Adams, 1988. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335, 440–442.

26. A. Strasser, A.W. Harris, M.L. Bath, S. Cory, 1990. Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature 348, 331–3.

27. S. Cory, J.M. Adams, 2002. The Bcl2 family: regulators of the cellular life-or-death switch. Nature Reviews Cancer 2, 647–56.

CHAPTER 19

Postdoctoral Days at the MRC: My First Adventure in Structural Biology (1967–9)

Peter B. Moore

Department of Chemistry, Yale University, New Haven, CT

Figure 19.1. Peter Moore.

Several months ago, it was decreed that my office would be moved to a room about half the size of the one it had occupied for many years. This diktat forced me to sort through a lifetime’s accumulation of books, notebooks, paper and memorabilia in an effort to decide what to throw out. One of the treasures unearthed during that winnowing was a three-ring notebook with a green cover that bears the inscription, ‘Laboratory Data. Medical Research Council, Laboratory of Molecular Biology, Cambridge’.

About the same time, I received an email message from Hugh Huxley inviting me to write an essay about the eighteen months I spent with him as a postdoc at the LMB (October 1967–April 1969) for a book project he was contemplating. I was flattered to be asked, and I would have agreed in any case, but my enthusiasm for the project was bolstered by the discovery of this ancient laboratory notebook. Only later, when I began reading the notebook, did I realise something I should have known all along. It describes the experiments I did at the LMB, but it does not explain why I did them, or what I thought I learned by doing them, which are the only two aspects of that work that anyone might find interesting today, including me. Thus, the account that follows depends far more on my memory than I had hoped it would.

For the record, I got my PhD in biophysics from Harvard in 1966, under the direction of James D. Watson. My thesis describes some experiments I did to elucidate the mechanism of messenger RNA binding to the ribosome. From February 1966 to the early autumn of 1967, I was a postdoctoral fellow at the Institut de Biologie Moléculaire at the University of Geneva, where I worked on the purification and characterisation of the ribosomal proteins from E. coli with Alfred Tissières.

Jim Watson visited Geneva once or twice while I was there, and during one of those visits he advised me not to return to the United States until I had spent some time at the LMB. (In those days, most of us referred to it as the MRC, but LMB seems to be the preferred usage today.) His advice made sense because the LMB was obviously the world’s premier centre for both molecular and structural biology, and if you are Muslim, you have to go to Mecca. Jim arranged for me to join Hugh’s group as a postdoctoral fellow, and with some additional help from Jim, I obtained the fellowship needed to support this venture.

In September 1967, my wife and I loaded all our worldly goods into a VW, and left Geneva for Cambridge. As it happened, Alan Weeds, who had just obtained his degree at the LMB, owned a house north of town, more or less on the road to Ely, which he wanted to rent out because he was about to spend a year in the USA as a postdoc. Thus, for roughly a year we were tenants of Alan’s, and later we rented Ieuan Harris’s apartment on Cedar Court, while he also was on leave in the USA.

By the time I got to the LMB, the still quite new, but architecturally undistinguished building it occupied on Hills Road was full of people. As best I could tell then, it housed four classes of scientists: a (to me) surprisingly small number of predoctoral students; a much larger number of postdocs, many of them US citizens supported by US fellowships; a substantial cadre of mostly British post-postdoctoral scientists, whom I surmised had short-term appointments of one kind or another from the MRC; and a very small group of world-class scientists, who had permanent appointments. That last group, which provided the leadership for the LMB, included Perutz, Kendrew, Crick, Brenner, Sanger, Klug and Huxley.

Like many others who worked at the LMB in that era, I still think of its modus operandi as exemplifying the blueprint that all scientific research establishments should aspire to emulate. Pack the very best scientists you can find into a building, so densely that they cannot avoid talking to each other, and encourage them to interact in every other way you can. A canteen or dining room might be a good idea. (The facility itself need not be luxurious, and indeed, it is probably better if it is not.) Give those scientists ample staff support, and all the money they need to get on with the job. Stir well, and then be patient because good science takes time. My subsequent career has taught me that this recipe is much harder to execute than it is to describe. I still wonder how the MRC managed to do it so well for so long.

My move to Cambridge entailed a major change in scientific direction, as well as a change in location. Previously I had been doing biochemistry/molecular biology on the ribosome. As a member of Hugh’s group I would be working on muscle, a system about which I knew nothing. (In retrospect, it is remarkable that Hugh agreed to take me on. What was he thinking?) Not surprisingly, and as the aforementioned notebook confirms, my first months at the LMB were spent learning the rudiments of the muscle trade, including how to prepare G- and F-actin, as well as myosin and its various proteolytic fragments, and the complexes these molecules form with each other.

I soon discovered that muscle biochemistry is not for the faint of heart. After I had been in residence for a while, we ran out of the dried muscle powder used for actin preparations, and a large white rabbit was procured to provide the muscle needed to replenish our supply. It was decided that the rabbit should be sacrificed in a way that would not upset it emotionally because it was believed that muscle obtained from an animal that had met its maker in a peaceful state of mind was superior to that obtained from an animal that had been terrified at the time of its demise. In hindsight we probably should have offered the rabbit a tasty bit of lettuce (‘Here bunny, bunny’), and when it bent over to eat, hit it smartly on the back of its head with a blunt object, but we decided instead to kill the rabbit by overdosing it with ether in a covered wastebasket. I will spare you the details, but I can assure you that the rabbit objected to these proceedings as strenuously as it could, for as long as it could, which was a very long time indeed.

Once that nasty bit of work had come to an end, we removed the striated muscles from the rabbit’s body. It then had to be shredded in a blender in the presence of, in this case, litres of cold acetone, and following filtration, the resulting acetone-soaked gunk had to be air-dried on the laboratory bench. The motor of the blender arced visibly as it ran, and if there was a hood in the room, which there may not have been, its flow rate was inadequate. For a long time that afternoon it was not obvious that we would get the job done before we were asphyxiated by acetone fumes, or blown to kingdom come when they ignited, or both. I am happy to report that all hands survived (except the rabbit), but note that in later years, had something similar taken place in my own laboratory, I would have been sent to jail. O tempore, o mores, as the poet once said.

I have no recollection of having ever discussed with Hugh the direction of the research I was to do. It may have happened, and I may have forgotten. However I have long suspected that he intended from the outset that I do exactly what I wound up doing. I was given a desk in a very small office that was already occupied by Brian Clark of tRNA fame, Jean Witz, a French postdoc who was interested in viruses, and David DeRosier, an American postdoc working for Aaron Klug, who was in the midst of determining the structure of the tail of bacteriophage T4 by three-dimensional reconstruction starting with EM images. The procedures David was using to solve the structure of the T4 tail could be applied to any biological assembly that has helical symmetry, and as Hugh and Aaron knew, muscle is full of such things. My job was to find out what could be learned if the technology David and Aaron had invented was applied to EM images of muscle fibres of various sorts, including the complexes that form between fibrous actin and fragments of myosin.

It turns out that there were very good reasons for wanting to know as much about the structures of these assemblies as one could. If memory serves, it was during the time I spent at the MRC that it finally became unmistakably clear that the contractility displayed by muscle does not derive from its remarkably regular, three-dimensional structure, as some had thought. Instead, in the presence of ATP, a single molecule of myosin that is associated with a single actin fibre can do mechanical work. Most eukaryotic cells, including many that have nothing to do with muscle at all, contain both actin and myosin, and use them to move things around. Striated muscle is simply an array of such elementary units, organised so that the forces they exert all add.

At the outset, I had no knowledge of any of the things I needed to know to do this work. I have already mentioned the gaps in my education in the area of muscle structure, physiology and biochemistry, but there were other lacunae that were no less critical. For example, I knew nothing about crystallography and Fourier transforms, and the DeRosier/Klug approach to reconstruction goes through reciprocal space. That gap was filled using a copy of Fourier Transforms and X-ray Diffraction, a small book written by Lipson and Taylor that I still have in my collection. In addition, despite the existence of all the computer programs David had produced to solve the T4 problem, a lot of programming still needed to be done, and I had never written a line of code in my life. David gave me a pamphlet that had a title along the lines of ‘Fortran for the Hopelessly Ignorant’, and before long I was submitting jobs to the IBM 360 /44 that was housed in the Institute of Theoretical Astronomy. (As I recall the capacity of the memory of this monster was 45 kB. A tear comes to the eye!)

The decks of IBM cards we generated were shipped to the computer twice a day, Monday through Friday, and twice a day those cards, plus whatever output had resulted when the computer read them, returned to Hills Road. If the pile of fanfold printout that returned was thin, you knew your submission had been a disaster, and it was back to the drawing board. If it was thick, you felt a sense of satisfaction just looking at it, even if it turned out to be less than everything you wanted, which was usually the case. Like everyone else who did hard time computing in those early days, I have a rich supply of colourful computing stories that I tell my younger colleagues when I want to impress them with how hard things were back in the old days, when men were men, etc., etc. Suffice it to say that it takes a long time to debug a program when you can only submit twice a day.

I have no idea how computing costs were managed by the LMB in that era, but I do know that its junior scientists treated time on the IBM 360 /44 as though it was a free good. This enabled us to do whatever we thought it might take to solve our problems, unburdened by concern that financial ruin might ensue if one of our larger computations went awry, which they often did. Having subsequently had to operate in environments where hourly charges were levied for computer use, I can assure you that the LMB model is the one to use if the objective is to get science done; accountants be damned.

The last piece of the puzzle was electron microscopy. If memory serves, there were two electron microscopes at the LMB when I got there, both Siemens Elmiskops. Every aspect of the operation of an Elmiskop was controlled manually, and while they were adequate optically, I soon realised that you had to be really good (or really lucky) to get useful micrographs from them. Every time you used one, you had to adjust its optics to correct for astigmatism, which could be a challenge, and image focus was always a problem for me. Shortly after I got to Cambridge, Hugh began teaching me how to run these instruments. In principle, it wasn’t hard to do, but I never did it very well. The fraction of the micrographs I produced of negatively stained F-actin, actin decorated with myosin S1, etc. that were worth keeping was disappointingly small. I was (and still am) in awe of people like Hugh and John Finch who could walk up to one of those microscopes, insert a specimen into it, twiddle the knobs, and emerge a few minutes later with a publication-grade image. My batting average did not improve much after the installation of a new Philips microscope, which occurred about a year after I got to Cambridge, even though it was much easier to operate. Many of the micrographs used for the reconstructions we did were obtained from Hugh’s remarkable collection.

In the end, the work we did on recovering three-dimensional models for muscle protein assemblies from EM images succeeded well enough to convince others that the game was worth playing, but our results were far from definitive. F-actin, and decorated F-actin, the two assemblies we concentrated on, are not ideal objects to study using the helical reconstruction methods we employed. F-actin is a flexible, loosely wound, two-stranded rope. Its helical pitch can vary along its length, and EM images of straight segments that are long enough to include several helical repeats were/are hard to come by. Many reconstructions of both structures have been published by others since 1969, some obtained using techniques that do not require specimens to display perfect helical symmetry. In addition, most of the specimens studied in recent years have been embedded in vitreous ice rather than being surrounded by negative stain. These reconstructions have much higher resolutions than we could ever have obtained using 1968 technology, and they are superior in every other respect. (It would be a sad commentary on the reconstruction field if the structures produced when it was in its infancy could not be improved on four decades later!). However, these early reconstructions did show the tilted and slewed mode of attachment of each individual myosin S1 monomer to each of the actin monomers, and did show the elongated and curved shape of the S1 itself, all features which later work has confirmed. The structure represented the attachment of the myosin cross-bridge to actin at one step of the crossbridge cycle, probably the end of the working stroke.

I left the LMB to take up a faculty position at Yale in April 1969. Even though Hugh told me that he would support me if I wanted to stay on, which was an attractive offer, after more than three years abroad I was ready to go home. For reasons that are irrelevant here, my return to the USA was accompanied by a return of the focus of my research to the ribosome, where it stayed, effectively, for the rest of my career.

It might be argued that my return to the ribosome somehow implies that my time at the LMB was wasted, but that is not the way I see it. The eighteen months I spent at the LMB had a huge positive impact on me. In the first place my experiences there taught me something that was profoundly important (to me), namely that I liked doing structural biology much more than I liked doing the biochemistry/molecular biology I had done first at Harvard and then at the University of Geneva. In addition, my experiences at the LMB gave me a solid, albeit unconventional foundation in structural biology that has stood me in good stead.

My stay at the LMB had other, less obviously scientific, benefits. In that era, its American postdocs socialised a lot, and as a result my wife and I got to know many of them well: Joan and Tom Steitz, whom we had both known previously at Harvard, the DeRosiers, the Abelsons, the Dahlbergs, and many others. (I beg the indulgence of those I have not named.) Those friendships have endured. In addition, I got to know the other young people who worked with Hugh in that era, e.g. John Haselgrove, Nigel Unwin, Jim Spudich and Gary Borisy, and as an individual who worked between Aaron’s group and Hugh’s, I got to know some of Aaron’s people as well: David, of course, but also Tony Crowther and Linda Amos. I cannot remember how it was that I got to know Richard Henderson, who subsequently came to Yale as a postdoc, and Keith Moffat. Both were then graduate students working on crystallographic problems. However, I distinctly remember riding down Hills Road on the bus one afternoon talking with Keith about time-resolved structure determination, a then pie-in-the-sky idea that he subsequently took very seriously. For the most part, our paths diverged in the years that followed, but it is still true that whenever I meet one of these people, I sense the existence of a bond that derives from shared experiences in Cambridge.

The record shows that many of the young scientists who were part of the LMB community in my era went on to have fine careers. It is hard to know whether this is because the LMB only admitted capable young scientists to its precincts, or because time spent at the LMB made young scientists capable. At the very least, the LMB can take pride in having done no harm! But I would argue that it did something positive for all of us that was quite basic. It showed us what good science is, and how it gets done.

Around 2002, not long after Tom Steitz’s group solved the crystal structure of the large ribosomal subunit from Haloarcula marismortui in collaboration with my own, I gave a seminar at the LMB on ribosome structure. It was the first time in years that I had set foot in the place, but there were many familiar faces in the audience. After the talk, Max Perutz, who had been listening intently, approached me to ask some detailed questions that he must have thought inappropriate for the larger audience. I had barely known Max when I was at the LMB; he had been the lab director, whom I had regarded with a respect that bordered on awe, and I had been a postdoc working in a different area. At one point in the conversation I tried to tell him that what we had done with the ribosome was no more than what he had taught us to do. I am not sure the words came out quite that way, but what I have written here conveys my intent. Everyone who has ever solved the structure of a macromolecule owes Max and the LMB a lot.

From a scientific point of view, and many others, the eighteen months I spent at the LMB were arguably the most productive months of my life. Hugh’s invitation to write this essay gives me the opportunity both to thank him in public for having made it possible, and to pay homage to a remarkable institution.

CHAPTER 20

Ribosome Profiling 1960s’ Style (1967–70)

Joan Argetsinger Steitz

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT

Figure 20.1. Mark Bretscher, Christine Roberts, Jeff Roberts and Joan Steitz.

Figure 20.2. Tom Steitz and Mark Bretscher.

Photos: Mark Bretscher

I did postdoctoral work at the LMB because of its excellence in protein crystallography. I am not a crystallographer – my husband is. But the LMB was also producing exciting advances in the molecular genetics of gene expression. My thesis mentor at Harvard, Jim Watson, had written to his friend Francis Crick saying that I would be arriving with my husband, Tom Steitz, who was to join David Blow in the LMB Division of Structural Studies to decipher the catalytic mechanism of chymotrypsin. Francis agreed that I could work in the LMB Division of Molecular Genetics, headed by Sydney Brenner and himself, perhaps in part because I had met him over a year earlier when he had visited Harvard. Jim had suggested to me and my three female graduate student roommates that Francis might enjoy ‘a respite from faculty wife dinners’. We had written Francis an invitation to dine with us on pink perfumed stationery. He accepted and entertained us for hours with stories that only Francis could tell.

Tom and I arrived at the LMB in late November 1967. I was armed with a National Science Foundation (NSF) postdoctoral fellowship – all the American postdocs, who made up the majority of the non-staff scientists in the lab, brought their own salary support from independent sources. Space was very tight as the lab was just about to undergo a two-fold expansion. Francis therefore suggested that I start with a theoretical project in the library. I knew that experiments, not theory, were my forte and arranged to talk with all the permanent staff members in the division about giving me a bit of bench space. I decided to accept the offer of Mark Bretscher, who was working in the area I had proposed in my NSF application – the initiation of protein synthesis – and had been recommended highly by Wally Gilbert, who interacted closely with the Watson group at Harvard. Specifically, Mark was testing the idea that ribosomes find the correct start site on an mRNA by scanning from the end; his experiments elegantly demonstrated that ribosomes could initiate even on a circular message (Bretscher, 1969, Journal of Molecular Biology 42:595), disproving this hypothesis.

My next decision was what specific project I should undertake. An exceptionally challenging experiment was mentioned by several staff members and postdocs. The idea was to isolate fragments from a bacteriophage genomic mRNA that were bound by ribosomes under conditions of the initiation of protein synthesis and then determine their nucleotide sequences using the new methods for RNA sequencing that Fred Sanger and his colleagues upstairs in the Division of Protein and Nucleic Acid Chemistry had recently developed. At that time, little more than the terminal nucleotides of bacteriophage genomic mRNAs had been determined. Although many of my recently arrived postdoctoral colleagues (all male) had considered taking on this project, they rejected it as too risky. They needed solid results to present on the US job market in 2–3 years’ time.

I, on the other hand, having already worked in several labs both in the US and Germany, had never seen a woman professor or lab head. I therefore had no illusions that I would ever have a ‘real job’; my future career would be as a research associate in someone else’s lab. Why not attempt a risky project? If successful, it would represent a quantum leap in our understanding of mRNA structure in general and specifically would tell us what differentiates an initiator AUG from the many in- and out-of-frame AUGs that must exist in every message. Moreover, I had worked with the bacteriophage R17 genomic mRNA as a graduate student. Although the gene order was not yet determined, it was known to encode three proteins – the A protein (a minor but essential constituent of the bacteriophage particle), the major coat protein and the viral synthetase (which produced both the minus template strands and the progeny RNA plus strands during bacteriophage replication). Thus, I might be lucky and manage to analyse three start sites at the beginnings of the open reading frames representing the three genes.

All the tools to carry out my project were available in the open lab system provided by the second and third floors of the LMB. I knew how to produce 32P-labelled R17 RNA and earlier studies in several LMB labs had worked out conditions for generating initiation complexes using E. coli ribosomes without subsequent elongation of polypeptide chains. Further, I was able to exploit a recently-published methodology (Takanami, Yan and Jukes, 1965, Journal of Molecular Biology 12:761) for digesting away exposed portions of the mRNA using RNase A. Mark Bretscher’s advice and encouragement were invaluable on all aspects of the experiment.

I ran into trouble at the step where I tried to isolate the ribosome-protected mRNA fragments that should represent the initiator regions. After months of column chromatography, plagued by problems such as fraction collectors stopping in the middle of the night and horrendous losses of the radioactive fragments on the vast surface areas provided by columns, I nearly gave up. A year had passed since we had arrived at the LMB, and I had no mRNA fragments that I could even begin to analyse. I spoke with Sydney Brenner about alternative projects I might undertake. Just before the Christmas and New Year holidays in 1968, I decided that the next experiment would be my last, but I would attempt to separate the ribosome-bound fragments from the vast excess of digested 32P-RNA using a sucrose gradient instead of column chromatography. Sydney had encouraged me with the advice that science could sometimes be like a bad marriage: ‘You make one last try before giving up for good.’

Analysis of the gradient revealed a peak of radioactive fragments sedimenting with the ribosomes, while the vast majority of 32P-RNA was at the top. When I subjected the peak to RNase digestion and ran a Sanger fingerprint on the isolated RNAs, I knew that I had material to sequence from the moment the X-ray film emerged from the processor! With the help of Bart Barrell and Fred Sanger and of others from both the Molecular Genetics and the Protein and Nucleic Acid Chemistry divisions who were newly-honed in the art of RNA sequencing, I analysed the spots from the fingerprint and then pieced them together. This revealed that I had obtained fragments corresponding to the beginnings of each of the three R17 genes: central AUGs were succeeded by triplets corresponding to the known N-terminal protein sequences of the three R17-encoded proteins. Everyone was suddenly interested in my results. There were clearly non-coding nucleotides between genes, but no specific sequence seemed to be the obvious signal that directed ribosomes to attach and start there (Steitz, 1969, Nature 224:957). Only somewhat later did it become clear that the polypurine stretches appearing upstream of all three initiator AUGs were important. Six years later (Steitz and Jakes, 1975, PNAS, 72:4734), I would demonstrate that these purine stretches base pair with the pyrimidine-rich 3′-end of the 16S RNA in the small ribosomal subunit – the so-called Shine-Dalgarno interaction, as it later came to be known.

One day Mark Bretscher came to me and asked: ‘How would you like to be a guinea pig?’ Curious, I responded positively. He explained that, as a resident fellow of Caius College (established 1348), he would like to put me up for ‘member of the room’. A member of the room would have rights to dine at High Table with the dons, use the Senior Common Room and have access to certain other college functions. To Mark’s knowledge, no woman had ever been appointed to college membership, as this was prior to any of the Cambridge colleges becoming co-educational. (Caius finally accepted undergraduate women only in 1979.) Mark explained that he would use an unassailable argument to support my nomination. Specifically, I was a visitor at the lab and would only be a member – if elected – temporarily. But, the college could then claim the precedent of having elected a woman member before any other Cambridge college. He ventured that the proposal had little chance of success, but was worth a try. I said yes, and somehow Mark’s persuasive powers prevailed with the other dons.

Arranging to dine at Caius College was a formidable process. One had to ring the head butler and announce one’s desire to dine at least a day in advance. Thus, the psychological barrier was substantial. Of course, on those several occasions when I did manage to summon up courage and arrange to dine at High Table, I was the sole woman. Usually I was seated next to the Master, Joseph Needham. He was an embryologist and biochemist, and also a renowned expert on Chinese science and civilisation. It was difficult, but not impossible, to maintain a conversation with him. I also recall that, at that time, Stephen Hawking, who was a fellow of Caius, was only moderately debilitated by the disease that would rob him of both his mobility and his speech, but not his brilliant mind; he could still make it up the stairs to the Senior Common Room with crutches. There were two other perks associated with my college membership. One was access to the Gonville and Caius wine cellar. I recall Tom’s delight in purchasing 1961 Lynch Bages for 30 shillings. This set a high bar for our lifelong indulgence in fine wines. The other perk was Tom’s ability to use the college’s grass tennis courts located close to the Crick apartments where we were living on Barton Road. Upon entering the courts, everyone was queried by the groundskeeper about being a member of the college. Tom simply responded that he was Dr Steitz. A check of the rolls revealed that a Dr Steitz was indeed a member of the room and since there had never been a woman member in Caius history, Tom was allowed on the courts – often with a guest.

My science at the LMB flourished because of the input of many others. The fact that the labs were open, rather than partitioned into small fiefdoms headed by permanent staff members, meant that there was constant interchange. Morning coffee and afternoon tea as well as lunch in the canteen were daily opportunities for sharing results and asking questions. Everyone mingled and interacted, and in my case not just with people having similar interests from the second and third floors. Occasionally Max Perutz would bring the banana and Swiss cheese that invariably comprised his lunch, along with the high stool that helped him cope with his painful back, to the table where I sat. Saturday morning coffee in the second-floor kitchen was ostensibly a further opportunity for scientific exchange, but was often dominated by a monologue from Sydney – on subjects as arcane as the Norwegian telephone book. There were only occasional interlab exchanges, but one series I recall distinctly was evening seminars on ongoing research at King’s College that brought us together with university researchers including such distinguished chemists as Dan Brown. Within the lab, the annual October meetings provided an overview by featuring the most exciting advances from each of the LMB divisions in a very concentrated format.

Not only the staff members at LMB, but our fellow postdocs were exceptional people as well as scientists. Friendships that were to last a lifetime were forged as we rubbed shoulders (literally) on a daily basis. Mark Bretscher also shared a bit of lab space with David Hirsh and with Jeff Roberts while I was at LMB. Other ‘visitors’ with whom I overlapped from both the second and third floors of the lab were John Abelson, Art Landy, Jim Dahlberg, Mary Osborn, Suzanne Cory, Jerry Adams and Harvey Lodish. When it became apparent that English antique furniture was pouring onto the market (because of a change in the tax laws), days in the lab became punctuated by trips to nearby auction houses and Mondays brought discussions of treasures that had been purchased on weekend excursions into the East Anglian countryside. The LMB was famous for its parties, where staff and foreign visitors mixed freely. Those at Mark Bretscher’s newly purchased 16th-century cottage in Swaffham Bulbeck were especially memorable. Each year in June, the May Balls provided a taste of the lighter side of English collegiate life.

Tom and I left Cambridge to return to the US in March 1970. He was to take up an assistant professor position at Berkeley in the department of biochemistry, an arrangement that had been made before we had gone to Cambridge in 1967. I was to work in the lab of Bruce Ames as a temporary postdoc; the Jane Coffin Childs Fund had awarded me a fellowship. We already knew that we would return to Cambridge for at least the summer since Tom’s grant application was not yet funded. Science-wise, it made much sense for us to return to the fertile research environment of the LMB. On our way across the US, we singly and together visited various universities that had invited us for seminars and ventured an interest in hiring us. This ultimately resulted in offers of assistant professorships for me, as well as Tom, from Princeton and Yale, but not from Berkeley. We decided to go to Yale, which has been our home ever since.

Trying to replicate the approach to science that I learned as a postdoc has inspired much of what I have done in my own lab over the years. But perhaps most importantly, the LMB – by nurturing and acknowledging important scientific contributions regardless of who had made them – built my confidence at a time that there were almost no role models for women in academic science.

CHAPTER 21

How My Career Was Shaped by the MRC-LMB (1967–70)

Thomas Steitz

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT

My three years of postdoctoral research at the MRC Laboratory of Molecular Biology in Cambridge between November 1967 and November 1970 were the most important time in the development of my future research direction and my perspective on how creative science should best be done. I learned that the development of new scientific ideas and the fun of science are greatly enhanced by constant discussions with others – students, postdocs and senior scientists in different, but related fields. I noted that a surprising number of the students and postdocs that I now see at Yale and elsewhere feel intimidated talking to more senior people in their field. In contrast, I felt that the research environment at the LMB in Cambridge, where I had the opportunity to talk to a large number of people whose excellence provided insights and goals for me to achieve, was very important to the development of my career.

After completing my PhD with Bill (‘the Colonel’) Lipscomb at Harvard, I spent three years at the MRC laboratory in the group of David Blow. He was recommended to me by Hilary Muirhead, who was at that time a postdoc with the Colonel and a former student with Max Perutz. On my first day in the LMB in Cambridge I ran into Max Perutz in the hallway where I said ‘Hello, Dr Perutz’, and introduced myself, whereupon he shook my hand and said, ‘Please, call me Max.’ This was in great contrast to other labs at that time and set the tone for the interactions I was about to experience with the early founders of molecular and structural biology, as well as postdocs and students.

In David’s lab I worked with a graduate student, Richard Henderson, on determining the X-ray crystal structures of chymotrypsin complexes with substrate analogues. Richard was a fabulous person with whom to work, both scientifically and as a fun person. We started by making a complex with formyl-tryptophan and examining difference electronic density maps, first at 5Å resolution and then at 2.7Å resolution. From this structure we could confirm and elaborate on the catalytic mechanism of peptide bond hydrolysis by chymotrypsin. We were also able to build a homology model of trypsin to propose how it recognises Arg or Lys side-chains in substrates and the source of its substrate specificity, which subsequent structures by other labs confirmed.

The Cambridge Laboratory of Molecular Biology was a completely unique and outstanding laboratory. It inspired and trained a very large group of postdocs from the US in molecular and structural biology who then returned and transformed these fields in the US. Virtually all of the postdocs were from the US and a very large fraction of them (about 40 per cent) are currently in the US National Academy of Sciences. Perhaps the most remarkable and unique feature of the laboratory was the canteen located on the top floor which provided coffee in the morning, lunch after midday and tea in the afternoon. The attraction was definitely not the ‘bangers’, the ‘toad in the hole’ or other culinary opportunities, but sitting down with a random collection of lab directors, postdocs and graduate students and talking about science. The canteen was set up by Max and run by his wife, Gisela. When I first arrived, it was so small that whenever I got through the food line, there were only a few empty seats. Consequently, I would have to sit at a table that might include Max, Francis Crick and Sydney Brenner as well as postdocs and students. Within about two months I had met nearly everyone in the whole laboratory. The conversations were always about science and experiments, never about the movie someone saw the previous night. Everyone contributed suggestions and/or criticisms. Initially I wondered how anyone got any experiments done since they were spending so much time in the canteen, and then I realised that the many discussions reduced the number of unwise or unnecessary experiments that were done and enhanced the good ones.

There were no weekly group meetings, but there was an annual one-week meeting for essentially everyone in the LMB which was generally known as ‘Crick week’. Francis would sit in the front row and frequently ask many questions. On one occasion I remember Fred Sanger presenting a talk on his recent research, and in the middle Francis jumped up and said, ‘Fred, if you did (this) and (this) and (this), then you would be able to find out (that) and (that).’ Fred, without taking his hand off the chalkboard, turned toward Francis and said, ‘That’s it, Francis, that’s it; you’ve got it,’ and then carried on. Since Crick week included a broad range of molecular and structural biology topics, it was very useful that directors in the front row would ask questions that many of us were reluctant to ask. During one lecture that involved a comparison of a process that occurs both in eukaryotes and prokaryotes, Max asked, ‘What is a eukaryote and what is a prokaryote?’, terms that were just beginning to be used. I was glad that Max asked the questions, since I had no idea what the terms meant. Sydney Brenner would have coffee available in his lab dishwashing kitchen on Saturday morning at about 10.30 am or so and would always be there with the postdocs and students from the molecular biology floor directed by himself and Francis, plus others of us who wanted to drop by. Sydney would invariably hold forth on some interesting topic with lots of funny stories and ‘one-liners’.

I learned about all of the major research problems being pursued at the LMB from Crick week, the canteen, Saturday coffee and the random conversations in the hall, as well as the quick evening trips to the pub for ‘last call’, which the American postdocs did. (The Brits were mostly not there at night.) It was at this time that I developed my interests in trying to understand the structural bases of the mechanisms by which the many proteins and nucleic acids that are involved in ‘Crick’s Central Dogma’ carry out their functions: how DNA is copied into DNA, DNA transcribed into RNA and finally the RNA translated to protein. This has been the focus of my laboratory’s research for the past 35 years.

Access to computing facilities was extremely limited at Cambridge. The LMB used the computer owned by the Cambridge University astronomy department; we were allowed to make only two submissions a day for the five working days, and the morning run could not take more than two minutes. I would check and recheck the computer cards I was submitting to eliminate as many mistakes as possible, because otherwise one of ten opportunities would be lost to me for the week. In retrospect, it is amazing that we were able to accomplish anything. Certainly, the timescale was longer.

At some point in my second year Brian Hartley, who was collaborating with David Blow on chymotrypsin studies, came up to me and asked what research project I planned to pursue when I left the LMB and began my own independent lab. I said I wanted to solve the structure of an aminoacyl-tRNA synthetase, ultimately complexed with substrates including tRNA. This is the step where a specific amino acid is attached to the tRNA containing the correct anticodon. Brian patted me on the back and said, ‘There, there, my boy. That is an interesting problem, but you must work on something you can actually do successfully. I suggest that you study the structure of hexokinase.’ I thanked him for the advice and ran down to the library to find out what hexokinase was. I subsequently learned by reading papers from Dan Koshland that the hexokinase reaction was his primary example of why some enzymes must undergo a conformational change upon binding the correct substrate, a process termed ‘induced fit’. He reasoned that if the enzyme was rigid with all of its catalytic groups properly oriented to catalyse the nucleophilic attack of the six-hydroxyl group of glucose on the alpha phosphate of ATP, then why would water not hydrolyse ATP in the absence of glucose? The six-hydroxyl group is after all a water molecule with some carbons attached. He hypothesised that the binding of glucose must cause a conformational change in the enzyme that is necessary for catalysis. I consequently started growing crystals of yeast hexokinase at Cambridge and subsequently spent the next ten years studying the various structural states of this enzyme at Yale (and ultimately showed that Dan was right). Mentorship is always essential, and not only from your direct supervisor. We eventually solved the structure of gln-tRNA synthase bound to tRNAgln and an ATP analogue, twenty years after my hallway conversation with Brian.

I submitted my first grant application to the National Institutes of Health (NIH) in 1969, I believe, in which I proposed to determine the structures of yeast hexokinase, of which I had managed to produce crystallographically suitable crystals. David had allowed me to obtain yeast hexokinase from Eric Barnard in Buffalo, grow crystals that diffracted to 2Å resolution and begin the search for heavy atom derivatives as a side project. At a site visit by an NIH panel I was asked by a panel member how I was going to collect the X-ray data. I said by using a diffractometer, whereupon I was told that data collection with a diffractometer would not work because the unit cell dimensions of my hexokinase crystals were too big (one dimension was about 200Å). This, of course, was a ridiculous comment, since one just moves the detector further away from the crystal, but the reviewer was firm and ultimately my first application was turned down. (A few decades later this reviewer asked me in an elevator while being escorted to my next faculty visit how we had solved the structure of hexokinase, and I said: ‘By using a diffractometer to collect data.’)

Some time at the end of 1969 or early 1970, Fred Richards was visiting the LMB, and I had an opportunity to talk to him. I asked him whether a faculty position for me was possible at Yale, and he said he would look into it and get back to me. Years later I learned from Sydney Brenner, while dining with him at King’s College, that Fred had run up to his office after talking to me and asked him if he could encourage another American postdoc in Sydney’s lab to either accept or reject the offer that he had received from Yale. Sydney immediately called the postdoc into his office, closed the door, put a piece of paper on the table and told the postdoc that he would not be allowed to leave the room until he had written and signed a letter to Fred either accepting or rejecting Yale’s offer. Sydney then took the letter rejecting Yale’s offer to Fred, who left Cambridge with two faculty slots in his pocket. Fred had wanted to have two positions available, since there were faculty in the department who wanted to hire Joan.

I went to Yale in the late autumn of 1970 and have continued my research there ever since. During the 1970s we worked on structural studies of hexokinase, as suggested by Brian Hartley, and then turned our attention to obtaining the structural basis of Crick’s ‘Central Dogma’ – DNA makes DNA, DNA makes RNA, and RNA makes proteins. Over the subsequent decades we have obtained the structures of the enzymes, factors, DNA and RNA captured in the various states of carrying out this process. Not surprisingly, I was inspired to pursue this research pathway primarily from my contacts with Francis while at Cambridge. As we worked our way through the pathway-DNA polymerase structures, RNA polymerase structures, CAP and aminoacyl tRNA synthetase, we got to the final step – protein synthesis by the ribosome. In 1995, we began to study the crystal structure of the 50S ribosomal subunit, and in 2000 we published its atomic structure to be followed almost immediately by Venki’s structure of the 30S ribosomal subunit. This was an exciting moment.

In the autumn of 1976 and spring of 1977, I took a sabbatical leave, first at the Max Planck Institute for Biophysics in Goettingen, Germany and then at the LMB in Cambridge: the contrasts between the research and interaction environments could not have been starker. In Goettingen I worked in the lab of Klaus Weber on purifying and crystallising the E. coli lac repressor. Klaus’s lab was great, but there was no interaction between different labs that mostly lived in separate towers. None of the directors had lunch in the dining hall. I never met the institute director, whose lab was one floor up and who often talked to Klaus in the hall. Upon my arrival in Cambridge, once again, I ran into Max in the hallway; he said we must have lunch together in the canteen so that he could catch up on what research I had been doing. In Cambridge I worked with Richard Henderson on using EM to study very thin, negatively stained crystals of lac repressor. I learned a lot about EM and have followed the field closely over the subsequent years, though I have not pursued it myself. High resolution cryo-EM is clearly having an increasing impact on structural biology, and the LMB has played an important role in its development.

What I learned from my years at Cambridge is the importance of ‘structured informal contact’; working in an environment where coffee, lunch and tea in the canteen, as well as contact in the hallway, lead to new ideas, problem solving and long-term, cross-field friendships.

Figure 21.1. Party at the Abelson cottage. Left to right: Mark Bretscher, Jean Thomas, Margaret Clark, Joan Steitz, Lynne Abelson, John Abelson, Brian Clark.
Photo: Thomas Steitz

CHAPTER 22

Becoming a Professional Scientist at the LMB (1967–70)

Paul M. Wassarman

Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY

In 2012 the Medical Research Council (MRC) Laboratory of Molecular Biology (LMB), on Hills Road in Cambridge, England, celebrated its 50th anniversary. Actually it was the 65th anniversary of the founding in 1947 of its predecessor, the MRC Unit for the Study of the Molecular Structure of Biological Systems at the Cavendish Laboratory, on Free School Lane in Cambridge. At the time the sole members of the unit were Max Perutz and John Kendrew who fifteen years later were to move to the new Hills Road facility and in the same year, 1962, share the Nobel Prize in Chemistry.

It has been 45 years since I arrived at the LMB as a postdoctoral fellow with John Kendrew and Herman Watson in the Division of Structural Studies. Somehow I had convinced the Helen Hay Whitney Foundation that they should support me and my family in Cambridge, despite the fact that I knew next to nothing about protein crystallography. The Whitney Foundation, and especially their executive secretary, Dora E. Young, were so very generous and kind to me and my family throughout my years as a postdoc. So with a fellowship from them I arrived at the LMB in July 1967 fresh from graduate studies in biochemistry at Brandeis University. It was my PhD supervisor, Nathan O. Kaplan, who suggested that I go to the LMB and learn something about protein crystallography if I was to continue as a protein chemist.

It was the first time I had been out of the States – in fact, it was the first time I had been more than 250 miles away from Boston and the first time I had flown in a plane. On the day we landed in England an American friend from Brandeis, Dick Hammerschlag, and I picked up a new Fiat 124 in London; it had been driven from Italy to England. Packed with me, my wife, our three children, and eleven pieces of luggage, I drove our new car slowly to Cambridge on the ‘wrong side of the road’. Upon arriving at our house on Queen Edith’s Way in mid-afternoon, we unloaded everything and I went off to the LMB on foot – it was less than ten minutes away from where we lived. I couldn’t wait to get started!

Figure 22.1. 1983 Symposium of MRC Fellows.

In front: Joan Steitz and David Hirsh.

Front row (left to right): John Smith, Steve Martin, Paul Wassarman, ———, Elsebet Lund, Jim Dahlberg, David Ish-Horowitz, Leslie Barnett.

Second row (left to right): Brian Clark, Gene Katz, Tom Steitz, Rita Fishpool (assistant), Martin Hooper, Art Landy, Tony Cashmore, Sydney Brenner.

Third row (left to right): ———, Sidney Chang, John Abelson, Hugh Robertson, Mike Mathews, Mark Bretscher, ———, Richard Pink, Malcolm Gefter.

Back row (left to right): Paul Sigler, Peter Rigby, John Kilmartin, Philip Rudland, ———, Martin Lubin, Keith Moffat, ———.

Photo: Paul Wassarman.

In July 1967 the LMB consisted of only one relatively small building at the new Addenbrooke’s Hospital site, about two miles from central Cambridge. I entered through the unlocked front door, was greeted courteously by someone in a small reception area to the right just off the entrance, and they phoned Herman Watson to tell him that I had arrived. I knew Herman since he had done a sabbatical in my department at Brandeis and lectured to us graduate students on protein crystallography. He arrived, welcomed me, and showed me my office, to be shared with two other people, and my lab bench, considerably smaller than I had had at Brandeis and to be shared with a technician, located on an upper floor of the building. I remember that along the way Herman introduced me to several people, including Max Perutz (who immediately said ‘call me Max’), Paul Sigler (with whom I listened to the 1967 World Series, Red Sox vs Cardinals, on Armed Forces Radio), Margaret Brown (Max’s secretary), Susan Coombs (John’s secretary), and others. I did not meet John; he probably wasn’t there. But then jet lag caught up with me and Herman and I walked back to Queen Edith’s Way, he greeted my family, and I went to bed exhausted shortly thereafter.

Although John Kendrew (made Sir John in 1974) was my sponsor, I must confess that we had very little contact during my time in Cambridge. In fact, shortly after arriving there I learned that he was rarely to be found at the LMB, so that meetings not involving him were often held in his ‘vacant’ office. John was rarely to be seen in the canteen, at seminars, or at other events during my years at the LMB, although I do remember him making a surprise appearance in the laboratory when the BBC arrived one day to produce a short film. Much of John’s time was spent serving on Ministry of Defence committees in London, and as editor-in-chief of the Journal of Molecular Biology, secretary-general of EMBO and EMBC, director of studies in Natural Sciences at Peterhouse, etc. We only spoke twice while I was a postdoc. We ‘chatted’ in his office for half an hour about a week after I arrived in Cambridge and this was repeated about one year later. We never discussed science. But this was not a liability since there were so many other outstanding scientists at the LMB with whom to interact and affiliations with a particular individual or group were completely flexible. Consequently, I’ve always been extremely grateful to John for bringing me into the LMB environment.

Thinking back, a few things surprised me upon arriving in Cambridge. Our house was heated primarily by portable electric heaters, there was no central heating, and a small coal boiler in the kitchen heated our hot water – a large supply of coal was stored just outside our back door. Generally, we could wash our dirty dishes or take a bath, but not both. Our refrigerator was about the size of a large breadbox, suitable for a couple of pints of milk and a dozen eggs, but not much else. It was expected that we keep our perishable food in the larder – a pantry adjoining the kitchen that depended on cold from the outside – cold was not readily available in July and August. But the house was completely surrounded by beautiful roses, red, yellow and white that lasted well into Christmas – unfortunately I was severely allergic to roses and had to receive shots from the local GP all winter long. Lastly, the water pipes were located on the exterior of our house so that when they froze in winter, and they did, we needed a plumber – the plumber explained that the pipes were outside so that they would be easier to get at when they froze – ‘not to worry’. After one year we moved into a modern centrally heated house on Almoners Avenue, also only a short walk to the LMB. No more pre-heating our bed with a hot water bottle in winter and having to choose whether to take a bath or wash the dishes.

What were my first impressions of the LMB? At first I was very surprised by the large number of American postdocs at the LMB. As I remember, in Structural Studies (headed by John Kendrew) alone there were several American postdocs, including Gary Borisy, David DeRosier, Harold Erickson, Bob Josephs, Peter Moore, Paul Sigler, Jim Spudich and Tom Steitz. In the two other divisions, Molecular Genetics (headed by Francis Crick and Sydney Brenner) and Protein and Nucleic Acid Chemistry (headed by Fred Sanger), there were many more American postdocs, including John Abelson, Jerry Adams, John Anderson, Jim Dahlberg, Malcolm Gefter, Howard Goodman, David Hirsh, Art Landy, Harvey Lodish, Dick Russell and Joan Steitz. I must say that I never really missed the States, probably because I was surrounded by so many Americans at the LMB – enough to play American football on the lawn just in front of the LMB; Max disapproved, but tolerated it. The Vietnam war was raging, anti-war protests took place everywhere, but somehow I felt insulated from the turmoil in the world at the LMB and in Cambridge. Research was the primary relevant issue that connected all of us and Cambridge was a perfect refuge from the unrest in the world.

Another aspect of the LMB that I remember well was the apparent democratic participation of everyone in the day-to-day life of the laboratory. There were no obvious barriers. All interactions were on a first-name basis. In retrospect, I’m sure there must have been conspiracies going on behind the scenes, but as a postdoc I was never aware of them. Doing science was exciting and enormous fun at the LMB, made considerably easier by Michael Fuller who seemed able to cater to the scientists’ every need for supplies, equipment, transportation into London, etc. These were wonderful times to be doing research at the LMB and to be living in Cambridge – despite the frequent rain and paucity of sunshine that was characteristic of the Fens.

Like many other American postdocs, one of the things I remember most about the LMB is the canteen. Initially it was relatively small and located on the top floor of the building; upon completion of the second LMB building it moved there. Virtually everyone, with the exception of John Kendrew, went to the canteen – junior and senior scientific staff, administrative staff, technicians, postdocs, students and visitors. No matter where you sat you could listen in on conversations going on at all of the other tables – particularly if Francis Crick was involved. It was crowded, the food was not particularly tasty (especially the ‘grey’ Brussels sprouts), but being in the canteen was an unforgettable experience. Three times a day, for coffee in the morning, lunch at midday and tea in the afternoon almost everyone would gather in this small space, in part for food, but primarily for conversation, mainly about science. In the canteen you got to know everyone, learned about all the research going on at the LMB, and were entertained by, among others, current and future Nobel laureates, Fellows of the Royal Society, and Members of the National Academy of Sciences. Life in the canteen was democratic. You heard about the latest breakthroughs in molecular biology almost as soon as they occurred. What an experience. What an education. When American postdocs returned to the States they frequently reminisced especially about the LMB canteen and in several instances tried to replicate it at their institution. However, administrators never seemed to understand or appreciate the relevance of a canteen to research. Too bad.

Two of the annual events at the LMB were the in-house lectures given over a few consecutive days and the Christmas party. Almost everyone at the LMB attended the former and research continued at only a trickle during this period. The highlight of the latter was a skit put together by the postdocs and students. In 1968 I was persuaded by fellow postdoc Malcolm Gefter to play the part of Max Perutz in the skit with a plasticine scalp (in 1968 I had a full head of hair), a laboratory coat, and a few bananas protruding from my coat pockets. Just as I began my role, Max sat down directly in front of me; this was accompanied by a loud outburst of laughter from the audience. I’m sure that Max found my performance a bit rude – it was – but throughout my time at the LMB he and his wife, Gisela, were never anything but extremely thoughtful and kind to me and my family. As chairman of the governing board of the LMB (Max did not want to be called ‘Director’), Max believed that ‘it [the LMB] started with so many gifted people that I just had to let them develop and help them to get what they needed.’ I am certain that his benign, but very effective leadership and the outstanding example he set as a research scientist contributed enormously to the great success of the LMB.

For much of my time at the LMB I shared an office with Jens Birktoft and Tom Steitz, two terrific scientists and office-mates. At the time, who thought that Tom would become a Nobel laureate? We had a large window in the office for a time that faced Hills Road – but one morning the window was gone, replaced by concrete blocks, when the second LMB building was completed. Just across the hall from our office was a large room filled with desks and occupied primarily by graduate students, including Jonathan Greer, John Haselgrove, Richard Henderson, John Kilmartin, Keith Moffat and others. Further up the hall were small offices for Max Perutz, John Kendrew, Hugh Huxley, David Blow and Aaron Klug, as well as a larger office for Francis Crick, Sydney Brenner and John Smith. I remember that Uli Arndt, Joyce Cox, Tony Crowther, Bob Diamond, John Finch, Ken Holmes, Reuben Leberman (who turned out to be my cousin!), Hilary Muirhead, Herman Watson and others were just down the hall; as were all of the American postdocs in Structural Studies recalled above. No one had much space to call their own, but what an amazing group of outstanding colleagues to interact with.

One floor up from my office was my small lab bench where I worked with a superb technician, Jean Parkinson. The bench was located in a very large room occupied by many investigators from both Structural Studies and Molecular Genetics. As with the office space, no one had much room and we were pretty much on top of one another nearly all the time. I had much more bench space when I was a graduate student than when I was a postdoc. This sounds terrible, but it wasn’t. People like me working on crystalline proteins interacted constantly with others working on phage, viruses, bacteria, animal cells, worms etc. There was a hodgepodge of science going on all the time in the lab and the environment was incredibly stimulating, perhaps the most stimulating of any time in my career. Even out in the corridor, standing by scintillation counters waiting for the latest results, conversations about experiments were going on all the time. In this context, I should mention the different working styles of the British and American scientists at the LMB. The former seemed to spend far more time thinking about rather than doing experiments, and the latter worked round the clock at the bench. In the end, both approaches could be successful, but I learned that rushing headlong into experiments without much thought could be extremely wasteful of time, materials and effort. An important lesson.

For much of my time at the LMB I worked on glyceraldehyde-3-phosphate dehydrogenase (GPDH) crystallised from lobster muscle (L-GPDH). L-GPDH catalyses the sixth step of glycolysis in which glucose is broken down for energy and carbon molecules. The large crystals were yellow due to bound coenzyme (NAD+) and were quite beautiful. Each month or so I took delivery of two-to-three dozen Scottish lobsters which provided tail muscle for preparation of L-GPDH crystals and lots of other muscle tissue for many of my colleagues to enjoy at dinner. Prior work by Herman Watson and Len Banaszak had shown that the crystals of L-GPDH were orthorhombic with space group P212121 and that the unit cell contained four molecules of 140,000 molecular weight. I learned to mount and align crystals for X-ray diffraction, collect and process diffraction data, generate difference Patterson maps, and, in the end, at best became an ‘amateurish’ crystallographer. Although I published three papers on the chemistry of L-GPDH thiols while at the LMB (related to my attempts to make heavy atom isomorphous derivatives of L-GPDH), for several reasons, especially the large size of the unit cell, at the time we were unable to solve the structure of L-GPDH. So, I did not achieve the goal I had set for myself. However, it should be remembered that in the late 1960s the structures of only a handful of proteins had been solved and solving a structure was most often a long and relatively painful process.

On the other hand, without realising it, because of the superb scientific environment at the LMB, during my postdoc there I had acquired many of the tools necessary for a career as a research scientist. In particular, I had gone from thinking in two dimensions to thinking in three dimensions and this has served me well throughout my research career. I also learned that it is best to choose a novel research project and to focus on it intensely, rather than jumping from one project to another. Particularly valuable were my interactions outside of Structural Studies. From interactions with members of Molecular Genetics and Protein and Nucleic Acid Chemistry I became very familiar with various aspects of molecular biology and this also served me well over the years. Especially valuable were my many long conversations in the canteen with Sydney Brenner whom I idolised; perhaps inadvertently he greatly influenced the direction of my subsequent career. For within a couple of years after leaving the LMB I switched from structural biology to developmental biology as my primary research interest. I left structural biology because I never felt comfortable with the physics and computing involved in protein crystallography, and I didn’t enjoy playing a small role as a biochemist within a very large, multidisciplinary crystallography group. I wanted something more personal and developmental biology just seemed right to me.

In 1972 I moved to Harvard Medical School and began to work on certain aspects of mammalian (mouse) development – a far cry from protein crystallography. Again, it should be remembered that this was before molecular cloning, transgenic mice, knockout mice, DNA sequencing etc. came into being and transformed research on mammalian development. At about the same time I became friends with Mel DePamphilis at Harvard, a close friendship that continues today, and began a collaboration with him on SV40 chromatin replication and structure that lasted about a dozen years. By the late 1970s, thanks largely to my first graduate student at Harvard, Jeffrey Bleil, my own laboratory’s research became focused on the synthesis, structure and function of the zona pellucida (ZP), the extracellular coat that surrounds all mammalian eggs (see References below). The ZP plays important roles in oogenesis, fertilisation and preimplantation development. This focus continued for the next 30 years or so – at Harvard Medical School (1972–86), the Roche Institute of Molecular Biology (1986–96), and Mount Sinai Medical School (1996–2010) – and involved the participation of more than 50 graduate students, postdocs and research technicians. Many of my students and postdocs have gone on to successful careers in academia and industry, with several, including Richard Schultz, Harvey Florman, Eveline Litscher, Monica Vazquez, Chengyu Liu, Zev Williams, Huayu Qi and Luca Jovine continuing to work on various aspects of mammalian development in their own labs. Others, including Jeffrey Greve, Ross Kinloch, Richard Roller, Michael Cusick, Sergio Lira, Rosario Perona, and Costel Darie have gone on to successful careers in other areas of research. One of the most gratifying aspects of 40 years of research has been the creativity, collegiality and friendship of the people who participated in it with me.

Looking back, it is clear to me that my experiences at the LMB had a profound and lasting influence on my research career. When I arrived there in 1967, PhD in hand, I was a well trained biochemist, but a relatively immature research scientist. Immersion in the rich research environment at the LMB and, most significantly, daily exposure to the collection of outstanding scientists who worked there at that time, enabled me to become a professional scientist. It was an enormous privilege to be a postdoc at the LMB in the late 1960s and today I remain extremely grateful for having had the experience.

REFERENCES

Wassarman, P.M., December 1988. Fertilization in mammals. Scientific American 255, 78–84.

Wassarman, P.M., 1988. Zona pellucida glycoproteins. Annual Review Biochemistry 57, 415–42.

Wassarman, P.M., 1999. Mammalian fertilization: Molecular aspects of gamete adhesion, exocytosis, and fusion. Cell 96, 175–83.

Jovine, L., C.C. Darie, E.S. Litscher, and P.M. Wassarman, 2005. Zona pellucida domain proteins. Annual Review Biochemistry 74, 83–114.

Wassarman, P.M., 2008. Zona pellucida glycoproteins. Journal of Biological Chemistry 283, 24285–9.

CHAPTER 23

A Memorable Year: Physarum actin comes to Cambridge, Laboratory of H.E. Huxley at the LMB, 1968–9

Vivianne T. Nachmias

Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA

Figure 23.1. Vivianne Nachmias.

I will try to recount for this memoir the remarkably favourable events that made my year in Dr Hugh Huxley’s laboratory 44 years ago so interesting scientifically and for me, so memorable.

But first I need to explain how this happened. It would not have occurred were it not for the prestigious Phillips lectures at Haverford College. In 1966, Dr Huxley came to give one of these lectures. I had just joined the Department of Biology, where I was continuing to study the filaments and fibrils that I had recently discovered in amoebae while a postdoc at the University of Pennsylvania. Dr Huxley talked with the biologists before giving his lecture. I showed him some of my electron micrographs of fibrils in fixed and sectioned amoebae and he told me of the value of uranyl acetate for ‘negative staining’. Especially for small specimens that could be closely surrounded by the electron dense film, the ‘negative stain’ method provided excellent resolution of details of structure (Huxley and Zubay, 1960). This technique, I thought, might help me prove that the filaments I had discovered were not the favourite candidate of that period for production of cytoplasmic streaming, i.e. the recently discovered microtubules, as the visiting committee on my proposed grant strongly believed.

The source of cytoplasmic streaming had been a puzzle for many years. Noburo Kamiya’s notable work over many years on the plasmodium of Physarum polycephalum indicated that some kind of complex arrangement of pressure flow was one source of the oscillating cytoplasmic flow (cf. recently: Kamiya, 1988). Amoebae move without the oscillation. Both A. proteus and the giant multinucleated amoeba, Chaos chaos, show marked streaming; put out rounded pseudopodia while retracting a tail or ‘uroid’ region as they move about on surfaces. They modify this motility and change shape dramatically when they surround and ingest prey. A long search in the literature for proposed mechanisms showed that it was a rich time for hypotheses ranging from jet propulsion, to some action of the newly discovered microtubules. A brief, early, prescient theoretical paper by Ariel Loewy (1949), however, emphasised the likelihood that some kind of contractility related to the mechanism of muscle contraction is the most likely source of such fluid movements. Searching for structures that could underlie these movements, the only hints I could find in electron micrographs of conventionally fixed (osmium) C. chaos were a few faint lines in an occasional uroid – not seen elsewhere. To stimulate stronger ‘contraction’, I tried stimulating the amoebae with a cationic dye that binds firmly to the extensive anionic surface. When cells are so treated in the cold, then washed and warmed, they develop a greatly enhanced convoluted uroid where the dye collects as they begin to move. Since the osmium may penetrate the ‘coat’ slowly, I gently ‘pricked’ the surface of the recovering cells with single hairs to allow faster fixation.

Amazingly, in such maltreated amoebae, I found remarkable swaths of thin filaments, both in networks and in parallel arrays ~7–8 nM in diameter and some several micra long. There were also areas of 15 nM diameter filaments that measured less than a micron long and seemed solid and not similar to microtubules (Nachmias 1964, 1968). The two types were most often separate.

These structures were very exciting and yet puzzling as the work of Huxley (1963) showed clearly that muscle contraction depended on the interaction of two kinds of filaments. Yet, many images in the amoebae appeared to show intermediate forms between these two types of filaments, and when fully formed, there were no obvious interdigitations between them. What, then, were they? The thinking at that time was that the contractile filaments of muscle represented unique proteins, as each cell type in multicellular animals was thought to synthesise its own protein suitable for its role in the body.

So how best to find out more about these structures and their relation to streaming? Try to purify the filaments? Chaos is very poor material for harvesting proteins, as it is watery, hard to grow in large amounts, and lives on live Paramecia, offering potential contamination.

Luckily at Haverford College, which had just acquired an electron microscope and was happy to have another operator, I could try negative staining. Also, the new assistant professor was Dietrich Kessler, who was interested in the mitochondria of the slime mould Physarum polycephalum. This engaging, if ambivalent creature can live on raw old-fashioned oats, as our chair, Ariel Loewy, had found earlier in his pioneering work on a viscous extract that responded to ATP. It is not hard to obtain ~50 grams a week, starting with an inoculum from the sterile shaker flask cultures Dr Kessler used for his work. First, however, I continued to work with Chaos, and using negative staining, found that the thick, short filaments in Chaos, when centrifuged down and negatively stained, appeared dense throughout and hence were definitively not microtubular as the stain would have penetrated the interior (Nachmias, 1968), so that debate seemed resolved. The ends of some of the short, thick filaments appeared to fray out into very fine strands, again suggesting that they were somehow formed from the finer filaments. I considered using Physarum to try to purify similar filamentous material. Then there came a very fortunate event: the chance of a sabbatical. My husband wanted to go to Cambridge, England to do psychophysics. I wondered if I could visit the laboratory of Dr Huxley there and somehow study filaments of streaming cells.

The very good news was that I could. With a leave of absence from Haverford, I found a welcome in Dr Huxley’s lab and began to try to grow Physarum from sclerotia. It was heavy going; the mould was for some reason recalcitrant and did not flourish. Dr Brenner suggested early ageing, which happens with such cultures. It seemed discouraging, but then, another favorable event was that my colleague at Haverford, Dr Kessler, offered to send extracts. Dr Huxley had proposed a very interesting experiment: to see whether filaments from Physarum could react with muscle myosin from a vertebrate. A long shot because of the evolutionary separation for these organisms! Dr Kessler would make filament preparations from Physarum using Hatano and Oosawa’s procedure (1966) and send them to London by overnight air freight. As I recall, Dr Huxley’s response to ‘how to get them fast?’ was succinct: ‘But it’s dead easy to get to Heathrow, just go straight down the M1 and go down to the freight depot.’ (I think it was M1 then; it is now M11 on Google maps and about 51 miles). So I did. The first trip was of course the most exciting. To meet the night flight, it was best to leave Cambridge at about 4.30 am. Favourably, the night sky was full of stars and there were very few automobiles on the road so I could concentrate on staying to the left. I just remember that all went fine, the plane was on time and the thermos had not broken. There was still ice in the bottom.

There were indeed long filaments in the preparation, about 80Å wide, but not ‘clean’: many had some material over the filaments with an occasional fine ‘hair’ emerging from the main filament. Much of this material could be removed with a ‘wash’ including ATP. Now the real test began: a fragment of muscle myosin’s ‘head’, known as subfragment 1 (HMMS1), prepared by a method of Susan Lowey and colleagues, was being made by Alan Weeds, a coauthor. He provided a sample. It was remarkable to see that a drop of appropriately diluted S1 from the mammalian muscle made a very clear angled complex with the filaments from the strange and ancient Physarum very similar to its interaction with muscle actin.

Dietz sent better preparations but they still were not very pure. Serendipitously, he then obtained permission from Mark Adelman and Ed Taylor to try their then-unpublished method (1969) for column purified Physarum actin. The first good preparation showed fine clean filaments, often bundled, and from these various clear images the S1 treatment resulted in many regular arrowheads so that one could measure the crossovers carefully. It took some time to obtain really good micrographs, using the ‘holey grid’ method devised in Dr Huxley’s lab. In this method, portions of the filaments were stretched over holes with no support or interfering background material, again using uranyl acetate for the negative staining. In one set of measurements we found the ‘decoration’ to have an average axial periodicity of 363Å compared with muscle thin filaments similarly treated at 350Å. ATP would remove the muscle S1 as it does with mammalian actin.

There were a few difficulties: at one point in late winter some drilling was going on in a nearby part of the building, and I could not get good pictures. Hugh was unfazed – he suggested that perhaps the operator felt the drilling but the microscope was not affected. And indeed good resolution soon returned and arrowheads with 36 nm repeats were seen (Nachmias et al, 1968). It was even possible to get decent optical diffractions showing helicity (not shown).

This specific, reversible interaction between these two proteins over a great distance in the evolutionary tree seems remarkable even now. That it would have the same periodicity and angle as muscle actin interacting with HMM-S1 seems to underline the great conservatism in this essential step in actomyosin-based motility. We hypothesised at the time that actin has to interact with at least three molecules, two with itself (or three) and one with myosin, and this would tend to minimise changes; in fact it has been found to interact with a large number of other molecules – more other proteins than any other molecule (Erickson, 2007). Actin is remarkably stable over time: Hightower and Meaghan (1986) compared gene stability measured as a replacement of nucleotides affecting amino acids as 1 per cent per MY for globin and 1 per cent per 30–244 MY for actins overall. It has recently also been suggested that internal interactions of domains in the molecule may help to keep actin conserved in sequence and so in structure (Galkin et al, 2002).

Shortly after our publication, Ishikawa et al (1969) showed that many cell types in cultures made from chick had fine filaments that formed similar arrowheads with HMM actin; some of these cells were not expected to contain actin. So the ‘decoration’ turned out to be a useful test to detect an actin filament. And as to the amoeba story, Pollard and Korn in 1971 confirmed the probable interpretation of fine filaments in amoebae (A. proteus) as actin, showing that filaments formed in vitro under special conditions reacted with muscle heavy meromyosin to form quite regular arrowhead structures (one can conclude that it would be also true for Chaos chaos). A year later, I was able to obtain fairly pure preparations of Physarum myosin and to show that it formed arrowhead structures with muscle actin (Figure 6 in Nachmias, 1972a), and later that the myosin formed short thick filaments in vitro, about 0.5 microns long, like the thicker fibrils seen in Chaos (1972b).

In the 44 years since our work with Physarum, many remarkable discoveries have shown other properties of its myosin; in addition a great family of myosins has emerged and an amazing array of proteins that interact with actin, fitting the implication of its conserved structure. Control of the formation and character of actin filaments and actin pools and potential roles of polymerisation and depolymerization are examples. Yet we still do not understand cytoplasmic streaming. In the amoebae, lacking the ruffled anterior margin of tissue culture cells, what are the roles of the filaments that are now clearly actin and myosin? New approaches will hopefully clarify this difficult problem.

In retrospect, a major result of my year in Dr Huxley’s laboratory at the LMB was a realisation that Darwin’s ‘deep time’ is necessary to try to grasp for how long these two molecules, myosin and actin, have been working together to allow cells to move about, and so to live. I feel very lucky to have been there at that time and to have participated in the success of this pioneering hybridisation experiment.

REFERENCES

M.R. Adelman and E.W. Taylor, 1969. Further purification and characterization of slime mold myosin and slime mold actin. Biochemistry 8, 4976–88.

H. P. Erickson, 2007. Evolution of the cytoskeleton. BioEssays 29, 668–77.

V.E. Galkin, M.S. VanLoock, A. Orlova and E.H. Egelman, 2002. A new internal mode in F-actin helps explain the remarkable evolutionary conservation of actin’s sequence and structure. Current Biology 12, 570–75.

S. Hatano and F. Oosawa, 1966. Extraction of an actin-like protein from the plasmodium of a myxomycete and its interaction with myosin A from rabbit striated muscle, Journal of Cell Physiology 48, 197-202.

R.C. Hightower and R.B. Meagher, 1986. The molecular evolution of actin. Genetics 114, 315–32.

H.E. Huxley, 1963. Electron Microscope Studies on the Structure of Natural and Synthetic Protein Filaments from Striated Muscle. Journal of Molecular Biology 7, 281–306.

H.E. Huxley and G. Zubay, 1960. Electron Microscope Observations on the structure of Microsomal Particles from Escherichia Coli. Journal of Molecular Biology 2, 10–18.

H. Ishikawa, R. Bischoff and H. Holtzer, 1969. Formation of Arrowhead Complexes with Heavy Meromyosin in a Variety of Cell Types. Journal of Cell Biology 43, 312–28.

N. Kamiya, R.D. Allen and Y. Yoshimoto, 1988. Dynamic Organization of Physarum plasmodium. Cell Motility and the Cytoskeleton 10, 107–116.

A.G. Loewy, 1949. A Theory of Protoplasmic Streaming. Proceedings of the American Philosophical Society 91, 326–9.

V.T. Nachmias, 1964. Fibrillar Structures in the Cytoplasm of Chaos chaos. Journal of Cell Biology 23, 183–88.

V.T. Nachmias, 1968. Further Electron Microscope Studies on Fibrillar Organization of the Ground Cytoplasm of Chaos chaos. Journal of Cell Biology 38, 40–50.

V.T. Nachmias, H.E. Huxley and D. Kessler, 1970. Electron Microscope Observations on Actomyosin and Actin Preparations from Physarum polycephalum, and on their Interaction with Heavy Meromyosin Subfragment I from Muscle Myosin. Journal of Molecular Biology 60, 83–90.

V.T. Nachmias, 1972a. Electron microscope observations on myosin from Physarum polycephalum. Journal of Cell Biology 52, 648–63.

V. Nachmias, 1972b. Filament formation by purified Physarum myosin. Proceedings of the National Academy of Science 69, 2011–14.

T.D. Pollard and E.D. Korn, 1971. Filaments of Amoeba proteus II. Binding of Heavy Meromyosin by Thin Filaments in Motile Cytoplasmic Extracts. Journal of Cell Biology 48, 216–219.

CHAPTER 24

The Lifelong Influence of Max Perutz and the LMB (1968–71)

Michael Levitt

Department of Structural Biology, School of Medicine, Stanford University, Stanford, CA

Figure 24.1. Michael Levitt, c.1968.

In the winter of 1967, aged nineteen and in my final year of a BSc degree in physics at King’s College in London, I applied to do my PhD at the LMB but was told that they had no place for additional PhD students. On the advice of a college friend, who went on to become a super-successful businessman, I wrote back saying that if I could not come in 1967, what about 1968? This obstinacy got me to the next level and I was asked to come to LMB for an interview. There I met John Kendrew, Max Perutz and other members of the Structural Studies division. I thought the interviews went well, but the outcome was less satisfactory: I was asked to wait for a year, after which they would decide if they could take me. Again my friend intervened and advised me to go to Cambridge unannounced to try to resolve the situation. Thus, I drove from London to Cambridge in April 1967 – it was a Friday, a beautiful spring day blessed by a dazzling yellow sun in the sky balanced by bright daffodils all around the lab – waited outside Max Perutz’s office and approached him as soon as he came out. I quickly said that my concentration and my ability to study for the upcoming final exams were being affected by the uncertainty about my PhD future. Max listened sympathetically and said that he could do nothing without consulting his co-division director, John Kendrew. At this point I had the good sense to thank him profusely and leave quickly with the faint hope of hearing back from them.

To my utter amazement, my persistence paid off and when I telephoned John Kendrew’s secretary on the following Monday, I was told that they had decided to definitely accept me as a PhD student in 1968 and that they would help me find something to do during the intervening year. Kendrew felt strongly that I should spend the year with Shneior Lifson at the Weizmann Institute in Israel but my plans were more ambitious, involving spending time at research centres in Paris, Boston and California rather than in a small Middle Eastern country. In retrospect it seems that my audacity knew no bounds, swinging wildly from the slim chance of being accepted to outrageous plans for the intermediate year. John was much wiser and did not budge. He did, however, sweeten my visit to Israel by getting me a Royal Society exchange fellowship that was meant for postdoctoral exchange; as it was in its first year, there were no other candidates.

My year in Israel had two highlights. One was that I started to work in the brand-new field of computational structural biology as a programmer for Arieh Warshel and Shneior Lifson, writing the first program for simulation of protein motion by energy minimisation. The other was that I met my wife, Rina, and we married in August 1968 just before leaving for Cambridge.

Thus it came to pass that I joined the lab as a PhD student in September 1968 on the same day that Nigel Unwin joined as a postdoc – I well remember our chat in the original cafeteria that was above the old library. Shortly afterwards, there were the annual lab lectures and I was so excited to attend. My strongest memory is an odd one. A second-year PhD student, Peter Gilbert, was giving a talk on his model of the cerebellum and Francis Crick, whom I had not yet met, told him: ‘Peter, please explain this very slowly and simply. Explain it so that Max can understand.’ I was shocked by this disrespect for the head of the entire lab but no one else seemed concerned, especially not Max. I met Francis a few weeks later in another stroke of unexpected luck. A close friend of Rina’s had met Francis Crick in September 1968 when he was visiting Israel with Leslie and Alice Orgel. She had asked Francis to take some books back to Cambridge as a gift for Rina and so we were invited to his home, known as the Golden Helix, for Sunday tea. This was an incredible honour and it led to me being involved with a model for the structure of tRNA that had featured so prominently in the lab talks. It also led to me getting to know and work with Aaron Klug, the fourth of the towering heroes who so influenced my science. As a fellow South African who was a decade younger than Max, John and Francis, Aaron seemed more approachable. He was a team player who liked to work with a larger group that he directed.

MY SCIENTIFIC INTERESTS AND LMB

The influence of LMB on my scientific interests is stronger now than it was in 1968. As I wrote above, John Kendrew sent me to Israel to work with Shneior Lifson and specifically to learn how to use potential energy functions and massive computer simulations to calculate the properties of macromolecules of biological interest. Kendrew knew what he was doing and Lifson and his student Arieh Warshel were great teachers. Thus, I arrived at Cambridge having written the first program to move the atoms of a protein by minimising an empirically calibrated potential energy function. Having this under my belt gave me the freedom to explore science broadly as I worked towards my PhD. In addition to modelling tRNA with Francis and Aaron, I worked in the newly invented field of computational structural biology. My PhD thesis, entitled ‘Conformation Analysis of Proteins’, dealt with the empirical energy functions that I was introduced to in Israel. It described the basic form of the energy function, different coordinate systems and energy minimisation, before introducing a method for deriving a potential energy function that stabilised the known X-ray structure of a protein molecule. It then used this potential to energy-refine protein structures by moving atoms so that they fit stereochemical laws, to interpret problematic regions of electron density maps, to examine the transition state in the enzyme action of lysozyme and to examine the tertiary structure changes occurring in haemoglobin.

This early work set the stage for most of what I, and indeed much of the field, have been doing since then. In the last 45 years, computing has become at least 10 million times less expensive, computers now dominate all walks of life and biology has become information-rich. These developments naturally had a profound influence on my science as well as on my personal life. Specifically, our initial simulation of molecular movement by energy minimisation morphed into simulation of movement by Newton’s second law as computers allowed hundreds, thousands, millions and billions of iterations. The same methods could be applied equally to proteins, RNA and DNA and they could also be used to refine structures against NMR and X-ray data. I still use computers to explore molecular motions and flexibility and now, if anything, I am even more involved in the traditional LMB approach to structure. For example, one aspect of my current work focuses on the combined use of mass spectrometry, cryo-electron microscopy and R-value exploration of X-ray data with computer methods that allow large macromolecular complexes to be solved with less information. We have tested this on eukaryotic chaperonin, a large 950 kilodalton complex that is built from eight different but similar gene products and which was hard to solve by conventional methods due to quasi-degeneracy. In collaboration with Roger Kornberg, these methods are also being applied to transcription (Pre-Initiation Complex and Mediator). We are also developing new methods to simulate motion of these large systems combining natural moves, normal modes, analytical soft bodies and stochastic simulation. Both projects are relatively new and will keep me busy for the next decade. In my personal life, computers – especially email – have liberated me from the bonds of space and time. They have compensated for a weak memory, poor spelling and so many other ‘disabilities’.

INFLUENCES FROM MY TOWERING HEROES OF SCIENCE: MAX, JOHN, FRANCIS AND AARON

Beyond the very direct influence on my scientific career, propelled by John Kendrew’s good sense that made me a pioneer in computational structural biology, I was enormously influenced by the personalities and working habits of Max, John, Francis and Aaron. In planning this essay, I have come to realise how much I am a product of these early interactions. Thus, I would like to describe my ‘Lessons of LMB’ and illustrate them with anecdotes that have remained with me all these years. In doing so, I am only too aware that memory is not a factual recording of the past, but an edited personal version of what happened which is designed to help one relate to the present and future rather than portray the past with any accuracy. In compiling these lessons, it became clear that Max Perutz had influenced me more than anyone else. I have published papers with Max, Francis and Aaron, worked most closely with Aaron Klug and spent two years as Francis Crick’s only postdoctoral fellow shortly after he moved to the Salk Institute in 1977. Max Perutz’s influence on me has been like a fine wine, a little strange at first but maturing over 50 years to be a guiding light to me and, I imagine, to countless others.

THE LESSONS OF LMB

General Lessons

As a student in the Division of Structural Studies from 1968 to 1972, I felt complete equality and total freedom. I even felt more important than the postdocs as there were fewer PhD students than postdocs; there were also fewer PhD students than Nobel Prize-winners. I was free to interact with whomever I wanted, and, to my retrospective regret, I ended up not publishing any paper with my PhD supervisor Bob Diamond; we did have adjacent and related papers in the 25 January 1974 issue of the Journal of Molecular Biology (JMB). As a student with a baby and a wife who had to work to support the family, I stayed at home a great deal: LMB showed a remarkable acceptance of ‘telecommuting’ at a time when the term had not yet been invented and the nearest home phone was a call box, five minutes’ walk away.

I interacted with each of my scientific heroes differently. Max accepted me into the lab with Kendrew and taught me more about scientific life than anyone else. John accepted me into the lab, and sent me to Israel where I learnt from Lifson and Warshel and married Rina. Francis involved me with tRNA, and I did a postdoc with him just after he moved to Salk, where we became close family friends through our wives, Odile and Rina, who sketched and painted together. Aaron worked closely with me on tRNA and chromatin. Our common South African roots and connections with Israel bound us. He is a wonderful colleague and I wish I had been able to work with him more.

Personal style

Max and Aaron taught me to be modest and deliberately self-effacing, to be disarmingly simple and act like the TV detective Columbo (played by Peter Falk). Basically, be super smart but do not show it. Francis could not be like this and his lessons would have been: be as smart as you can be, ask hard questions and have a method for thinking. His brilliant mind asked penetrating questions about my computer program that he knew nothing about. He feared nothing. Max taught me to be generous and empathetic to everyone, especially those who have least power and seem least important. When he won the Royal Medal from the Royal Society in 1971, he gave a party in his office for all technicians, workshop staff, photographers etc., and to which I was invited as a PhD student. We assembled and Max announced: ‘I went down to Fitzbillies [Bakery] on my bicycle and got these cakes and then brought some wine from college’, while we passed his gold medal around admiring its thickness and weight.

Management style

How to manage others is a very hard lesson to learn. Max did it by example and in an invisible manner. He controlled bureaucracy and prevented it from taking over by not ignoring it but rather by confronting it and winning. Aaron managed his scientific group by delegation, showing confidence in others. He empowered others by his interaction with them: it was as if he were an ‘idea ventriloquist’, giving ideas to others and making them think that they were their own. Essentially both Max and Aaron aimed to maximise what each person they managed could do. This simple rule is only possible to follow when you can efface your ego; as such it conflicts with the desire to control others that seems to drive most leaders.

Working habits

Max taught me to work for yourself, to work until the end, and to always communicate as well and carefully as you can. John taught me to care about scientific writing and to write clearly especially for the interested reader, saying: ‘Be fair to those readers who want to understand the details.’ As editor-in-chief of JMB, he took my first paper, covered the first three pages with red ink corrections and told me to do the same for the next ten pages. As a result I strive to write better with each passing year. All my heroes taught me to love science. Treat it as a vocation and develop the best scientific taste that you can. Francis taught me to mix working on hard problems with easy, ‘bread-and-butter’ problems. He also said: ‘Never be too busy to investigate a new and promising direction.’ Aaron taught me to read papers, and to annotate and remember them. We all remember his ceiling-high piles of papers that he could access randomly, at high speed and with near-perfect accuracy. He also taught me to never give up. In 1980, two years before winning his Nobel Prize, he had four papers rejected but he fought back and prevailed.

Recreational habits

Although Max, Francis and Aaron all made their families part of their scientific life, I never really appreciated the importance of real recreation until a decade ago. Looking back I now see that Max taught me to take your group on ski trips. The first for me was the Hirschegg (Austria) meeting in 1970. Max had a real love for the outdoors, especially skiing, climbing and hiking. In 1985 at Manfred Eigen’s winter seminar in Klosters (Switzerland), he showed that one could ski even if one’s back hurt so much that one could not sit down. Francis taught me to reward students with food, taking me to a feast at Churchill College in 1970. There, I remember talking to a lady in the proverbial ‘little black dress’. She asked me what I did in my spare time and I said that I hand-washed nappies for our nine-month-old baby. I felt good about doing this and thought it was funny but she turned away in disgust! An enduring memory of Max comes from an unusually warm sunny spring day in 1987 (perhaps exactly twenty years after I had confronted Max in 1967). At lunch, we all stayed on the balcony outside the lab canteen for an extra fifteen minutes enjoying the sun, but only Max put on his hiking boots and walked around Wandlebury and the Gog Magog Hills for four hours before I saw him return with well-muddied boots and a beaming smile. What a wonderful example of carpe diem!

Be different

Max taught me to be quirky and eccentric so people would remember me. He liked staying at people’s homes, and endeared himself to Rina with late-night requests for boiled potatoes and black bananas. He taught me to befriend children when he drove with us on a trip to the Anza Borrego desert. He sat in the back of the car talking to our children and suddenly asked us to stop as we passed a small store. He got out and returned with a small gift for each of our three sons. When he visited Stanford in 1989, he stayed with us again. He wanted to go for a walk and introduced me to the most amazing hill walk (the Dish), which I had ignored for the two previous years although I passed by it every day. I believe that Max rejoiced in being different. For years his back pain caused him to write standing up, and listen to seminars lying on the floor close to the lecturer’s feet. John taught me to have a secret life. I did not realise why he had had such a special connection to Israel and the Weizmann Institute until I read what is now clear for all to see in his Royal Society biographical memoir: he had wanted to go to Israel after the war to be with a young lady. I so wish I had known about this when he chaired a session at a meeting in Jerusalem in 1996 during which I took the opportunity to thank him profusely for his pivotal role in my career. Sadly, he was only embarrassed by my public display of gratitude.

Enduring wisdom

Max often said that there are no bad students, only bad teachers. This seemed so obvious that I looked it up online and found that many teachers disagree with the statement. Much wiser was Max’s desire to have his friends visit him before he died. As he said: ‘Come now instead of coming to the funeral’; this should be a lesson for us all. John, Francis and Aaron all used their connections to help me. In 1967, John got me a Royal Society postdoctoral fellowship in Israel. In 1969, Francis submitted my paper on a tRNA model to Nature and it appeared in print just 24 days later. In 1977, Aaron sent Rina and me to the prestigious Taniguichi Symposium in Japan, an amazingly exotic destination at that time. Over the years I have realised that mentoring and helping others is the one thing that gets progressively easier as you age. Not helping others means wasting the leverage that you have gained by simply growing older.

WHY IS LMB SO SPECIAL?

Was LMB so special because of the unique combination of personalities that were there? It is tempting to answer ‘yes’ and leave it at that. Based on my career working at different research institutes and academic institutions in the UK, Israel and the USA, I searched for something more. I believe that research at LMB is most like intramural research at the US National Institutes of Health in Bethesda, and both are different from university research. Let me try to be more specific by listing what LMB and the NIH labs I know best have in common. Both have great shared resources that are provided without requiring grants to individuals. Both have few PhD students, and small groups of less than three or four postdocs with no teaching required. Offices are tiny and people are forced into proximity with colleagues who are all first-rate. Most importantly, one treats administration as an unavoidable duty, does one’s scientific research oneself and continues on like this irrespective of age or seniority.

Stanford University is different from both places in that there are few shared resources and grant funding is essential. Groups are larger and teaching is required. This provides additional pressure that I have enjoyed, although I must question my sanity in saying this. At a major US research university one is bombarded by a multitude of outside influences that greatly enrich one’s life. For me, these have included a closer connection with medicine, engineering and basic sciences, with recent PhD students from computer science, applied mathematics, chemistry and physics. It has been wonderful to witness the transformation of Silicon Valley from companies involved in hardware (Intel, Apple, Silicon Graphics, HP), to the internet (Google and Yahoo), and to social networks (Facebook). Five years ago I discovered a hidden passion for physical activity and spend more and more time swimming, training in the gym and seriously hiking. The need to teach got me interested in online classes early on, and in 2003 I created a twenty-hour course on computational structural biology consisting of 600 two-minute ‘talklets’. The course gave itself for five years, was then opened to all and is still being used worldwide – as I discovered recently when our server went down. I still manage to write computer programs, but writing grants, having a larger group and being more involved in outside activities make it really hard to write papers alone. I have written just two sole-author papers in the past five years and this troubles me.

CONCLUDING THOUGHTS

As is so well known, Max’s maxim was: ‘In Science truth always wins’. This always seemed self-evident to me but as I have become more exposed to other fields of human discovery and persuasion, I see how rare and special it is for truth to win. A much crueller saying that I also remember from LMB is: ‘You are only as good as your next paper.’ Actually, given that one’s MRC salary is guaranteed until retirement, this seems less harsh than the US equivalent: ‘Your salary will depend on your next grant.’ Both sayings serve to illustrate that in science one is continuously judged, and this seems fair given how much freedom we scientists have. I feel that I have been living with my towering heroes of science for the past three weeks by reading biographies, reminiscing, talking to them and sending myself emails. I miss them so much!

CHAPTER 25

LMB memories: From the LMB to PKA (1969–70)

Susan Taylor

Department of Chemistry, Biochemistry and Pharmacology, University of California San Diego, La Jolla, CA

Figure 25.1. Susan Taylor.

My journey to the MRC Laboratory of Molecular Biology was unconventional. Unlike most of the other postdoctoral fellows from the United States at that time, I did not come with a background in protein chemistry, crystallography or molecular biology. I had no experience with proteins, DNA or RNA. I did not come from Harvard, Stanford or Yale. These were the more traditional paths that led postdoctoral fellows to the LMB at that time. My pedigree was quite different. I had done my PhD at Johns Hopkins University with a carbohydrate chemist, Edward Heath, working on the incorporation of lipids into lipopolysaccharide – unlikely credentials for the LMB. My destiny took me to Cambridge because I followed my husband, Palmer, who was joining the newly formed Laboratory of Molecular Pharmacology led by Arnold Burgen. My fascination with protein synthesis and tRNA synthetases through the recent advances in the initiation of protein synthesis and the importance of N-formyl methionine tRNA led me to write naïvely to Brian Hartley and Sydney Brenner. Given my background, I am still unclear why Brian invited me to join his laboratory, but this single event changed my life forever.

When I arrived at the LMB in January 1969, a whole new world opened up for me. At the time I was still considering the possibility of going to medical school upon our return to the States. However, once I was exposed to proteins and protein chemistry in a place where science and everyday life was integrated with crystallographers, molecular biologists and electron microscopists, I never looked back. I worked on a visionary project that Brian had initiated. It was the forerunner of genetic engineering that became routine later on. Brian’s dream was to evolve ribitol dehydrogenase in a chemostat so that it adapted to use a poor substrate, xylitol. While Peter Rigby was in charge of the chemostat, my task was to sequence the wild-type ribitol dehydrogenase. Although the project did not move forward quite as anticipated because the bacteria just kept making more and more of the wild-type enzyme and did not actually generate improved mutants until several years later, I learned to be a very good protein chemist. I spent most of my time separating peptides by paper electrophoresis in the large tanks across the hall from the DNA and RNA sequencing lab on the third floor. I learned to dansylate the peptides, and my hands were often blue from ninhydrin. I left knowing how to purify and sequence proteins, but there was so much more that I had learned – lessons that would have a lasting impact on the directions that my life would take.

Most importantly, I learned protein chemistry alongside crystallographers. David Blow became a close mentor and Tom and Joan Steitz became lifelong friends. The chymotrypsin structure was just solved so there was daily cross-talk between the Hartley and Blow labs. Max was always a strong supporter – especially in later years when I actually began to solve protein kinase structures in my own lab. Jens Birktoft was there and Stefan Magnusson, also in Brian’s lab, became a close family friend. From Stefan I also learned bird watching. Fred Sanger’s lab was just down the hall, and the focus at that time with Joan Steitz was on sequencing RNA. Brian’s new lab, where my bench was, was the first lab in the new wing. César Milstein’s and Ieuan Harris’s laboratories were just across the hall. Jonathan King, down the corridor in Aaron Klug’s lab, arrived with his movies of the penguins in Antarctica. Uli Laemmli was also there. On the other side of Brian’s old lab was Kjeld Marcker where Mike Mathews and Ole Westergaard were postdoctoral fellows. Jim Spudich was down on the first floor with Hugh Huxley. When I arrived, John Abelson had just returned to the States to join UCSD, the new fledgling university in San Diego. Many future friends such as Richard Henderson and Alan Fersht were over in the US as postdoctoral fellows. My later friend, Louise Johnson, was also in the US at Yale.

The LMB was an extraordinary place to do science. The new wing had just opened, including the expanded canteen on the fourth floor. At first I was surprised, like most US postdoctoral fellows, at the amount of time each day that was spent in the canteen – coffee at 11.00, lunch at 1.00 and tea at 4.00. However, the canteen was a wonderful place to learn science as discussions routinely ranged from protein sequencing and crystallography to protein synthesis and molecular biology depending on the seat that was available. I have never since been in a place that was so rich in science and so spontaneously interdisciplinary. I learned to think about science without boundaries and have tried ever since to replicate that unique environment in my own lab and in my own institution. It was never quite the same; never quite as intimate and never quite as intense.

I grew up in the Midwest – Wisconsin and Ohio – and then later went to graduate school at Johns Hopkins University in Maryland. Although I had travelled to Europe in the summer between my junior and senior years at the university, I had never lived abroad before, and being in Cambridge also changed my life and the lives of my children later on. Travel became routine and exploring new countries and new cultures has been a lasting adventure for me and later for my family. One of the great opportunities in science is the chance to meet scientists from other countries and to cross international boundaries. With its rich history around every corner, Cambridge was fascinating. It remains one of my favourite places in the world. We lived in an old house called Wytherton that was adjacent to the Gonville and Caius playing field, and its owner, Dr Joan Cooper, was a retired anaesthesiologist who remained a close friend until she died at the age of 102. It was before Sainsbury’s supermarket, and one had to shop on Saturdays and wait in each line – one for cheese, one for meat, one for eggs. It took a long time. Brussels sprouts were the major fresh vegetable for the entirety of the winter months. Teas at the Orchard in Grantchester were routine, as well as many trips to Mr Mortimer’s shop in Little Chesterford to browse around his wonderful antiques. This was a favourite hangout for many at the LMB and the Steitzs and Spudichs, like us, still have our daily lives enriched by the pieces that we brought back home with us when we left Cambridge. We spent many weekends driving around the countryside to favourite villages in the Cotswolds and to Lavenham. We often jumped on the train to go to London for a play or a concert. Going to Europe and crossing the North Sea to Bergen, Norway, expanded our horizons further. One summer we visited relatives in Czechoslovakia and then drove on through Yugoslavia to Greece, and then across to Italy. The next summer we went to East Africa, following in the footsteps of earlier travellers like Mike Mathews who had been there with VSO International.

The community of colleagues at the LMB opened so many new horizons for me, and when I was about to return to the States fate intervened again in the form of a seminar by Michael Rossmann, a former LMB member. Michael, now at Purdue, had just solved the structure of lactate dehydrogenase and was trying to sequence it from the structure data without much luck. At tea after his seminar we began talking, and I told him that we were about to return to the States to the University of California at San Diego, a new university that I knew little about. My husband, Palmer, had just accepted a job in the new Division of Pharmacology. I had no idea what I was going to do, but Michael did. He arranged for me to join Nate Kaplan’s lab where Nate and Bill Allison, another alumnus of the LMB, were supposed to be sequencing LDH. Obviously it was not high on their list of priorities, and so I joined Nate’s lab doing a third year of postdoctoral studies. My lab mates at that time were another postdoctoral fellow, Jack Dixon, and a graduate student, Craig Venter. So UCSD was also an exceptional place, although very different from Cambridge and still in its infancy. I moved on to a faculty position the next year and eventually got tenure mainly through my sequencing of LDH, which was a big deal at that time. As soon as I had pieced together 50 residues or so, I would fly to Purdue to fit the sequence into the LDH model that Michael had assembled in a Richards Box. It was an amazing way to learn about proteins, and Michael and I have remained lifelong friends. I think that I taught Michael about biochemistry and biology, so it was an enriching experience for both of us.

My experience with the LMB and Michael also primed me to move in new directions when Nate put a paper on my desk and said: ‘This is an interesting protein. You should think about working on it.’ And so I did. The paper from Fritz Lippman’s lab described the newly discovered regulatory subunit of cAMP-dependent protein kinase (PKA), and that fateful day led me on another journey that continues to this day. Had I not been trained around crystallographers, I would not have been so determined to solve the structure of this protein. Unlike most of the pharmacologists and biochemists working on PKA at the time, I knew that it would be essential to have a structure if we were ever to truly understand its function. So we began to purify proteins from pig heart and to identify active site residues by affinity labelling. Ten-kilogram preps with ten-litre columns were routine, as were trips to Farmer John’s in LA for pig hearts. It was a major task in those days. But the first protein kinase structure that we solved in 1991 expanded our understanding of this protein and the whole family by orders of magnitude, and we have continued ever since to define the molecular features that allow PKA and other kinases to function as dynamic molecular switches that regulate most of biology.

Although I never overlapped with Brian Matthews, Paul Sigler or Michael Rossmann, their stories about their days at the LMB added so much richness to my own legacy. They all became close friends – in part because we shared this common background. In thinking about the history of crystallography, I am also amazed at the vision that these early structural biologists had regarding public policy. We recently had a meeting at Cold Spring Harbor commemorating 40 years of the Protein Data Bank. The vision and dedication of these early crystallographers in insisting that data be deposited in a public resource was extraordinary and a great gift to the scientific community. Sharing knowledge was a fundamental principle.

CHAPTER 26

1969–71 at the MRC Laboratory of Molecular Biology in Cambridge: Pivotal Years in My Early Training

James Spudich

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA

I arrived in Cambridge on the train from London on a wintry day in January 1969. Snow covered the landscape. It was my first time in England, in fact my first time in Europe. I had just come from Kerala, where my wife Anna and one-year-old daughter Rani would be spending time with Anna’s family while I settled in Cambridge. I had not made previous arrangements about where I would stay that evening, so with baggage in tow, I booked a room in a pub just across from Cambridge train station. Experiencing an English pub was already a Cambridge adventure.

The next morning I got to the MRC Laboratory of Molecular Biology (LMB) at the end of Hills Road. At the front entrance of the LMB, I encountered a young woman who introduced herself as Linda Amos. I asked Linda where I might find Hugh Huxley, with whom I had come to work. She replied that I was most likely to find him in the basement at the rotating anode for collecting low angle X-ray diffraction data on muscle fibres. I was to look for ‘an elegant man with white hair’. Her description was perfect and Hugh was easy to find. He welcomed me to Cambridge, and after a brief discussion we agreed to meet the next day after I got settled.

I went to Lloyds Bank on Hills Road to establish an account, and was surprised that the bank manager came out of his office to meet me and struck up a friendly conversation. Hearing I was looking for a place to stay near the LMB, he called a friend who he thought might have a room to rent, and within the next hour I was in a comfortable second-floor room in the lovely home of a Cambridge mathematics professor. It was my first day in Cambridge, and it was a perfect start.

After settling in I walked over to the LMB in time for their famous 4.00 pm afternoon tea. There was an empty space along one of the large rectangular tables that dominated the space, and I sat next to a woman who introduced herself as Leslie Barnett. Leslie was a key scientist in Sydney Brenner’s laboratory. Realising that I had just arrived and knew no one in Cambridge, Leslie invited me to have dinner at her home, with her daughters Marion and Penny. Many such dinners followed, before and after Anna joined me in Cambridge later that year, and our friendship with Leslie and her family continued for more than 25 years until she passed away. Many such lasting friendships were the hallmark of those Cambridge years. Hugh and Frances Huxley have remained special friends. We catch up with them as often as possible, often in Woods Hole or when they visit California (Figure 26.1).

Figure 26.1. With Hugh Huxley at Brandeis in 1996.

We also keep up with Alan and Stella Weeds, another friendship from that period. They spent 1981 in California, when Alan was on sabbatical in my lab and worked on developing an in vitro motility assay using a barbed end actin capping protein that we had purified from Dictyostelium. The idea was to orient actin filaments on a surface to observe myosin-coated beads moving along them. We had not worked out good filament alignment as yet, and our first robust quantitative in vitro motility assay would have to wait another year.

Gary Borisy and Bob Josephs were fellow postdocs in Hugh’s lab, and Gary and I continue to connect on a regular basis. Other LMB postdocs at that time included Susan Taylor, David DeRosier, Peter Moore, Tom and Joan Steitz, Harold Erickson, Jon Weinzierl and Bob Sweet. All of our paths continue to cross and we always speak fondly of those years at the LMB.

While at the LMB, Peter Moore stimulated my long-standing interest in flying by taking me up in a glider at Cambridge airport one Saturday. It took another six years for me to take up flying in the US, and flying small single-engine planes has been my hobby ever since. For anyone who has been a pilot, Antoine de Saint Exupéry’s quote ‘I fly because it releases my mind from the tyranny of petty things’ resonates well.

Besides life in the laboratory, which was my main occupation during my Cambridge days, Anna and I managed to enjoy Cambridge for all its special qualities – walking along the Cam from our apartment on Barton Road, listening to music at King’s College Chapel, visiting all the other colleges and sites of old Cambridge history, having sherry at the Turk’s Head, creating a life-sized brass rubbing of Roger of Trumpington, and most exciting of all, riding the train from Cambridge to London to the theatre and then making a mad dash back to Kings Cross in time to catch the last train back to Cambridge. We remember seeing a young Ian McKellen at the theatre in Cambridge, going to the May Ball (see Figure 26.2), and seeing the first daffodils of spring covered with snow.

Figure 26.2. The May Ball in 1970.

For Anna and me, our time in Cambridge was a heady mixture of science, culture and all that goes with it. In addition our family grew by one member. Our daughter Serena was born in Cambridge and the endless stories she heard about Cambridge were the motivation for her to return to Cambridge, to Trinity College, for a term while doing her Masters degree at Stanford.

Working in the lab was all-consuming and a great pleasure for the two years that I was at the LMB. The scientific environment of the LMB was exceptional. There were stellar scientists to learn structural biology from! In addition to Hugh, Aaron Klug would play a significant role in the work I did there, and discussions with Max Perutz and other faculty and their lab members at afternoon tea was an absolutely essential part of the LMB experience. So many ideas and details of experiments emerged at the afternoon teas. A very significant difference between my LMB experience and my experience at Stanford, where I was a graduate student with Arthur Kornberg and a postdoc for one year with Charley Yanofsky, was that at the LMB postdocs were fairly independent colleagues. We chose our own research problems, and worked on them quite independently. There were fewer distractions at the LMB compared to Stanford, in part due to being fairly isolated out at the end of Hills Road, but perhaps also from the belief on the part of the LMB leadership and faculty that energies should be fully focused on creative research at the bench. Tea breaks and the scientific interactions they provided were much more important than a plethora of seminars, teaching activities, weekly group meetings and the like – as postdocs, we were allowed to just focus on our work in a very contemplative way.

It’s interesting to recall what brought me to the MRC-LMB. I was an undergraduate student at the University of Illinois, Champaign-Urbana from 1959 to 1963 majoring in chemistry. There a chance encounter with Woody Hastings launched my experimental science career in biochemistry and biophysics. Woody introduced me to oxidative reactions leading to bioluminescence, and interested me in energy transduction in biological systems, a problem I have worked on for most of my scientific career. Woody invited me to join his lab as an undergraduate research student. I set up distillation apparatus in his otherwise biology laboratory and synthesised various long chain fatty aldehydes using reduction of the corresponding long chain fatty acids, and studied the effects of the aldehyde chain length on the bioluminescent reaction of Vibrio fischeri luciferase. This reaction involves reduced flavin mononucleotide, oxygen, and long chain fatty aldehyde, and oxidation of the aldehyde was assumed to be important in the reaction. So I synthesised the α,β-unsaturated derivative of decanal, 2-decenal, which should oxidise much more slowly than decanal, and showed that it is a classic competitive inhibitor of the reaction. In the summers of 1962 and 1963 Woody took me to the Marine Biology Laboratory (MBL) in Woods Hole, where I helped him in the physiology course that he headed at that time. The energy and scientific camaraderie at the MBL was inspiring, and I was introduced to the breadth and the potential of biological systems during my summers at Woods Hole. High on that list was the question of how muscle transduced the chemical energy of ATP hydrolysis into mechanical movement, inspired by conversations with Andrew Szent-Györgyi, then teaching at Woods Hole.

In the autumn of 1963 I joined the PhD program in the new Department of Biochemistry at Stanford University, founded by Arthur Kornberg. One of the many remarkable aspects of the biochemistry department was that, while Arthur was my thesis advisor, Paul Berg, Bob Lehman, Dale Kaiser, Buzz Baldwin, Dave Hogness, and the new assistant professors Lubert Stryer and George Stark, were all my mentors. There I learned how important it is to reduce complex biological systems to their essential components and to create quantitative in vitro assays for the function of interest. I also gained the insight that interdisciplinary approaches were key to the understanding of biological processes, and thus I sought training in genetics and structural biology. I therefore spent one year at Stanford as a postdoc with Charley Yanofsky, working on the genetics of the E. coli tryptophan operon. One year with Charley immersed in molecular genetics served me well in my later work.

My earlier interest in energy transduction by muscle was rekindled when I asked Dale Kaiser where I should go to get training in structural biology, to prepare me for interdisciplinary approaches into experiments. Dale had just returned from a senior postdoctoral fellowship in Hugh Huxley’s laboratory, and he recommended LMB and Hugh’s lab highly. With Dale’s recommendation and Andrew Szent-Györgyi’s description of Hugh’s work in Woods Hole many years earlier, I wrote to Hugh and, happily for me, he asked me to join him at the MRC. That decision changed the path of my future scientific career.

When I joined Hugh at the beginning of 1969, he was just publishing his pivotal Science paper on the swinging crossbridge model of muscle contraction.1 My previous training was so focused on biochemistry and genetics that I remember being captivated by the power of the structural approaches Hugh used, both electron microscopy of fixed muscle and isolated proteins and low angle X-ray scattering of live muscle tissue, to reveal important concepts of how muscle works. From the perspective of my background, however, I was surprised that there was no biochemical in vitro reconstitution of the function of interest, movement. Furthermore, genetic approaches were largely missing. Both of these became focuses of my own laboratory after I left the LMB for my first faculty position at UCSF in the new Department of Biochemistry and Biophysics founded by Bill Rutter.

As a postdoc at the LMB, I was keen to contribute something new to Hugh’s lab and to the field. At the time, it was clear that the tropomyosin-troponin complex was important for Ca2+ regulation of muscle contraction, but the biochemical mechanism and structural aspects of the system were not worked out. No one was working on this at the LMB. John Kendrick-Jones (Jake) was a postdoc with Andrew Szent-Györgyi at the time at Brandeis University studying scallop myosin, which is Ca2+ regulated by direct interaction of Ca2+ with the scallop myosin light-chain binding domain. Setsuo Ebashi and colleagues in Japan showed that the tropomyosin-troponin complex was part of the thin actin-containing filaments in muscle,2 but the similarities of the small troponins to the myosin light chains led Jake and Andrew to propose that the troponins may be functioning in muscle by somehow physically interacting with the myosin, rather than only with actin filaments.3

It seemed to me that biochemistry of the system was needed to clarify this issue before I could do structural analyses. However actin preparations at the time were heavily contaminated by tropomyosin-troponin, and each component needed to be purified independently in order to quantitatively study their interaction with each other and with myosin. My first paper from LMB, published in 1971 with Susan Watt,4 a very talented technician working primarily with Alan Weeds at the time, described the actin and tropomyosin-troponin purification procedures we worked out. The actin purification method described in that paper is still cited extensively. The most important experiment in that 1971 paper was the biochemical titration that proved that the tropomyosin-troponin regulates the actin-myosin interaction by complexing with the actin, and in a 1:7 molar ratio of tropomyosin-troponin:actin. The amount of tropomyosin-troponin needed to inhibit the actin-activated myosin ATPase in the absence of Ca2+ was independent of the myosin concentration, but linearly dependent on the actin concentration (Figure 26.3, top). If Jake and Andrew’s interesting suggestion were correct, Susan and I would have seen a myosin concentration dependence in our experiments.

Since the biochemistry experiments made it clear that we had active actin-tropomyosin-troponin complexes, I began taking samples from those biochemical reaction mixtures and negatively staining them for structural studies using a Siemens Elmiskop IA transmission electron microscope (Figure 26.3, bottom). The Elmiskop IA allowed twelve pictures at a time on glass plates. After only twelve photos, one had to break the vacuum and process the plates in the dark room, examine them, and then reload with twelve fresh plates for another round! I still have the more than 1,000 photos I took in those days.

Figure 26.3. Interaction of tropomyosin-troponin with actin filaments. Top: A biochemical titration experiment of the amount of TmTn needed to inhibit the actin-activated myosin ATPase in the absence of Ca2+. The open and closed squares show that a sixfold increase in myosin concentration has no effect on the amount of TmTn needed. The other curves show that more TmTn is needed as the actin concentration is increased. Bottom: Structural studies of samples taken from reaction mixtures from the biochemical experiments. Pure actin filaments show a 36-nm repeat (first panel). Troponin repeats every 38 nm along the filament, as seen on individual filaments (second panel) or on magnesium paracrystals of actin-TmTn filaments (third panel). The 36-nm and 38-nm repeats are clearly seen in diffraction patterns of magnesium paracrystals (fourth panel).

It was the early days of helical reconstructions from electron microscope images, pioneered by Aaron Klug. A key investigator in Aaron’s laboratory, John Finch, helped me to learn the needed diffraction theory to analyse my images. I had the privilege of doing the second helical reconstruction from electron micrographs of actin filaments, following a reconstruction by Moore, Huxley and DeRosier of actin fully decorated with Subfragment 1 (S1) of the myosin molecule. The work I did with John Finch and Hugh5 defined the position of the tropomyosin-troponin complex along the long pitch grooves of the actin filaments at high Ca2+ concentration, where muscle contraction is turned on.

Our studies showed that in the absence of Ca2+, it was very difficult to decorate the actin-tropomyosin-troponin with S1; the S1 attachment was somehow blocked. My time in Cambridge was running out, and I was desperately trying to get a reconstruction of the complex in the absence of Ca2+ to see how the structure changed. Those reconstructions were being computer processed the night before the morning my family and I were to leave for San Francisco, where I had accepted a job at UCSF. Anna understood the importance of this result, and she suggested that if needed, she would leave ahead of me with our two daughters and I would change my ticket to follow in a few days. But the inverse Fourier analysis worked out at the last minute, and we left Cambridge together in an old-style black taxi that took all four of us all the way to Heathrow airport.

At UCSF, analysis of the reconstruction done in Cambridge in the absence of Ca2+ showed that the density of tropomyosin was moved away from the position seen in the presence of Ca2+, and some density appeared further out of the long-pitch grooves of the actin helix near the S1 binding site. But the density was weaker than I expected and it was only one experiment, so we didn’t include it in the Spudich, Huxley and Finch paper published in 1972. Nonetheless, that result coupled with the obvious blocking of S1 binding to the actin-tropomyosin-troponin filaments in the absence of Ca2+ led us to propose in that paper the steric blocking mechanism by tropomyosin-troponin. Those were exciting days and combining biochemistry and structural biology in such an interdisciplinary approach would define my general direction for years to come.

With training in chemistry, physics, biochemistry, genetics and structural biology, I set up my own laboratory at UCSF in 1971 in the new Department of Biochemistry and Biophysics. I chose to focus on two key questions in cell biology at that time; the molecular mechanism of how the chemical energy of ATP hydrolysis brings about mechanical movement by the myosin molecule, and what roles a myosin-like motor might play in non-muscle cells. To understand the molecular mechanism of energy transduction, the first and most important issue was to develop quantitative in vitro motility assays for myosin movement on actin. Of considerable interest, however, was to try to unravel the molecular basis of the myriad non-muscle cell movements apparent by light microscopy.

Between 1966 and 1969, Sadashi Hatano and colleagues6,7 in Japan and Mark Adelman and Ed Taylor8 in the United States had shown that actin and myosin are in the acellular slime mould Physarum polycephalum. Also in 1969, Howard Holtzer and his colleagues9 decorated actin filaments with myosin S1 in a variety of non-muscle cell types to give the characteristic arrowheaded appearance that Hugh had elegantly shown for the muscle proteins in 1963.10 Non-muscle cell motility was on the verge of an explosion of activity.

In the first year of my lab at UCSF we searched for an ideal model system to study cell movements. We explored Neurospora crassa, Saccharomyces cerevisiae, Physarum polycephalum, Dictyostelium discoideum, Nitella axillaris and other organisms, all unfamiliar to me at the time. Dictyostelium, a cellular slime mould, proved to be best suited for our purposes. Margaret Clarke, one of my first postdoctoral fellows, identified a Dictyostelium myosin with properties similar to conventional muscle myosin11 The year before, Tom Pollard and Ed Korn had described a lower molecular weight form of myosin in Acanthamoeba castellanii, which was the first different form of myosin found in non-muscle cells.12 This myosin came to be known as myosin I, initially named because it was single-headed, while the two-headed muscle-like myosin was called myosin II.

In 1977 I joined the new Department of Structural Biology at Stanford and years of work by my laboratory on Dictyostelium took advantage of a chance discovery by my student Arturo DeLozanne, that homologous recombination can be used to knock out genes in this organism.13 Arturo knocked out the single copy myosin II gene in Dictyostelium and provided the first genetic proof that myosin II is essential for cytokinesis, but is not required for cell migration. The latter was a surprising and important observation because up to that time it was assumed that myosin II drove the forward movement of cells. These successes along the way helped alleviate the frustration of not being able to establish a quantitative in vitro motility assay.

There were two possibilities for an in vitro motility assay that we explored for the decade 1971 to 1981 – put myosin on a glass slide and visualise actin filaments moving over them, or orient actin filaments on a glass slide and watch myosin move along them. We imagined coating 1-micron polystyrene beads with either actin or with myosin and visualising the movement of the beads, which were readily visible in the light microscope. Actin filament orientation was pivotal for measuring directed movement, whether we tried myosin-coated beads moving along an actin-coated slide or actin-coated beads moving on a myosin-coated slide. The best experimental system we developed at that time, which eventually worked, made use of our Dictyostelium barbed end actin capping protein, severin, that we identified and purified in 1981. Using biotinylated severin, we attached the actin filament barbed ends to an avidin-coated slide and flowed aqueous solution over them. Long filaments attached to the surface at one end would be expected to orient in the direction of the flowing solution (Figure 26.4a). We placed myosin-coated beads on these actin-coated slides and added ATP but saw only sporadic movements. In retrospect, we probably did not have sufficient alignment of filaments; we were not monitoring filament alignment at that time by electron microscopy, as we did later. In 1982 when Mike Sheetz came to my laboratory on sabbatical we returned to the Nitella system I had explored earlier and took advantage of the known orientation of actin filaments in Nitella to overcome the actin filament alignment problem. Peter Sargent, a neurobiologist in the structural biology department at that time, helped us cut open a Nitella cell, and we attached it to a surface to expose the actin fibers. We added myosin-coated beads and eureka! We saw robust ATP-dependent unidirectional movement along chloroplast rows, which mark the actin fibers.14

Armed with the Nitella results, Mike left my lab and went to the MBL in Woods Hole to explore whether myosin-coated vesicles may account for the particle movements observed in squid axons. Ron Vale, then a graduate student at Stanford with Eric Shooter, was fascinated by the movement of organelles in nerve axons and joined Mike at the MBL. To their great surprise, they found that movement in axons is not myosin-driven. Instead, they discovered the new molecular motor kinesin,15 a discovery that completely energised the field and opened up years of exciting work from their laboratories and many others.

Meanwhile, at Stanford, we were working to eliminate the vagaries of the complex Nitella substratum and establish a totally defined in vitro motility assay for myosin moving on actin. We returned to the biotinylated-severin actin-filament approach but now used electron microscope grids attached to the glass slides to monitor the orientation of the actin filaments under various buffer flow conditions (Figure 26.4a). My LMB training in structural biology came again to the fore. With well-oriented actin filaments, my graduate student Steve Kron and I achieved the long-term goal of observing ATP-dependent directed movement of purified myosin along purified actin filaments at rates that were consistent with the speeds of muscle contraction and other forms of cell motility.16 (Figure 26.4b.)

Figure 26.4. The first quantitative in vitro motility assay with purified actin and myosin. (a) The concept was to observe myosin-coated beads moving along fixed actin filaments, oriented by buffer flow. The actin filaments had biotinylated severin, a protein we identified and purified in 1981, bound to their barbed ends; the barbed ends were attached to an avidin-coated surface by way of the tight avidin-biotin link. (b) Myosin-coated beads move upstream toward the barbed end of surface-attached actin filaments. The position of each of the three bead aggregates is shown as a function of time. This was the first demonstration of quantitative, directed movement of myosin along actin with a totally reconstituted system. Taken from Spudich, Kron and Sheetz.16

Thanks to a report by Toshio Yanagida et al,17 we returned to the myosin-coated surface concept that we had considered earlier. In the 1970s, we could only consider monitoring movement by observing the translocation of beads to which the actin filaments were attached; there was no way to visualise the 9-nm-wide actin filaments directly. In 1984, Yanagida et al showed that individual actin filaments labeled with rhodamine phalloidin could be observed by fluorescence microscopy. Using Toshio’s observation, Steve Kron achieved robust ATP-dependent movement of fluorescent actin filaments on glass slides coated with purified myosin.18 This became known as the ‘Kron assay’.

The Kron assay was the robust quantitative in vitro assay for the function of interest that we had long sought. It opened the door for myriad experiments to examine how myosin works. Using this Kron assay postdoctoral fellow Yoko Toyoshima obtained one of the most significant results to come from my laboratory. She, working with Steve Kron and others in my lab, showed that the globular head, or subfragment 1 (S1) of myosin, is the motor domain.19 (Figure 26.5.) I think it’s fair to say that this observation brought back into view Hugh’s swinging crossbridge hypothesis, which was in some doubt between 1969 and the early 1980s for lack of sufficient direct evidence of changes in orientation of the head during the contractile cycle. Yoko’s observation eliminated competing theories to the swinging crossbridge hypothesis and focused research on the S1 head to understand how the myosin family of molecular motors work. Almost two decades of hard work led us to the early goals we had set for ourselves.

Figure 26.5. Subfragment 1 (S1) of myosin is the motor domain. We had previously shown that the heavy meromyosin (HMM) fragment of myosin could produce movement. In a pivotal experiment, Toyoshima et al19 demonstrated that all one needs for movement is the S1 head. The insert shows the S1 crystal structure obtained by Rayment and colleagues six years later.21

The combination of the in vitro motility assay and the Dictyostelium myosin null cell provided powerful tools for Kathy Ruppel, Taro Uyeda, Dietmar Manstein, William Shih, Coleen Murphy, Meg Titus, Tom Egelhoff and others in my lab to use mutations along the myosin molecule to define the biochemical, biophysical and assembly properties of the molecule. Our results were consistent with the proposed actin-activated myosin chemo-mechanical cycle largely derived from the elegant biochemical kinetic studies from Ed Taylor’s laboratory in the early 1970s.20 Then in 1993 Ivan Rayment and his colleagues obtained a high resolution crystal structure of myosin S1.21 (Figure 26.5.) Ivan’s pivotal work allowed us to place our mutational analyses in a myosin structure-function context.

Fundamental issues still remained – primarily to establish the step size that the myosin takes for each ATP hydrolysis, which was under considerable debate. A step size larger than ~10 nm would be inconsistent with Hugh’s swinging crossbridge model and one would be forced to consider alternate molecular mechanisms. It was critical therefore to observe a single myosin molecule go through one cycle of ATP hydrolysis and to measure the step size directly. This was achieved when my graduate student Jeff Finer and sabbatical visitor Bob Simmons modified the Kron assay by building a dual beam laser trap for single molecule analysis (Figure 26.6). Using the dual beam laser trap, a single actin filament was lowered onto a single myosin molecule and we were able to measure the step size as ~10 nm and the force produced at ~5 pN.22

Figure 26.6. In vitro motility taken to the single molecule level using the physics of laser trapping. Top: Two polystyrene beads attached to the ends of a single actin filament are trapped in space by laser beams. The filament is lowered onto a single myosin molecule on a bump on the surface. Bottom: Data obtained following the displacement of one of the polystyrene beads with time. Both the displacement and the nucleotide binding kinetics are obtained at low laser trap force. At high laser trap force one can obtain the intrinsic force of the motor.

Building the laser trap involved a collaboration between our group and the physicist Steve Chu. Our collaboration was highly unusual in that members of our labs physically moved to each other’s laboratory environments for extended periods of time. This was so productive for both labs that we convinced then Stanford provost Condoleezza Rice and our deans that such interdisciplinary exchange of students should occur broadly – between biology, physics, chemistry, computational sciences, engineering and clinical sciences. This led to our cofounding the global interdisciplinary Bio-X programme at Stanford, which I served as the first director. I went to London to bring our ideas to Norman Foster with the hope that he would be the architect for a new building at Stanford to serve as the hub of the Bio-X program. The Clark Center, named after a key donor, Jim Clark, has an excellent restaurant on the site where investigators gather for breakfast, lunch and even dinners, and there are rectangular tables to seat many people, just like those at the LMB! The layout of the LMB restaurant was perfect for enabling people to interact who may not encounter one another during their daily work. I explained the importance of the restaurant and how it was to be laid out to the Stanford president John Hennessey. When there was a hint that we might have to forgo the restaurant due to a shortfall of funds, I recalled my experiences at the LMB and how important it was to have a nice restaurant with rectangular tables that brought students, postdocs and faculty together in a casual setting for free exchange of ideas, LMB style! Hennessey understood and promised the additional funding to carry forward the now very popular restaurant in the Clark Center with the long rectangular tables!

Finally, one of my great satisfactions is that the more detailed understanding of energy transduction by myosin has led to potential clinical therapies. A small molecule that binds and activates β-cardiac myosin is now in clinical trials for the treatment of heart failure, and another small molecule currently in clinical trials activates skeletal muscle contraction and may aid patients with amyotropic lateral sclerosis and other diseases.

My training in structural biology at the LMB was pivotal for what we have accomplished over the last four decades. Those two years in Cambridge at the LMB with Hugh were inspiring and influential in the way I do science and interact with my young colleagues, and I remember those years with great fondness.

REFERENCES

1. H.E. Huxley, 1969. The mechanism of muscular contraction. Science 164, 1356–65.

2. I. Otsuki, T. Masaki, Y. Nomura and S. Ebashi, 1967. Periodic distribution of troponin along the thin filament. Journal of Biochemistry 61, 817–19.

3. J. Kendrick-Jones, W. Lehman and A.G. Szent-Györgyi, 1970. Regulation in molluscan muscles. Journal of Molecular Biology 54, 313–26.

4. J.A. Spudich and S. Watt, 1971. Regulation of skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. Journal of Biological Chemistry 246, 4866–71.

5. J.A. Spudich, H.E. Huxley and J. Finch, 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. Journal of Molecular Biology 72, 619–32.

6. S. Hatano and F. Oosawa, 1966. Isolation and characterization of plasmodium actin. Biochim Biophys Acta 127, 488–98.

7. S. Hatano and M. Tazawa, 1968. Isolation, purification and characterization of myosin B from myxomycete plasmodium. Biochimica et Biophysica Acta 154, 507–19.

8. M.R. Adelman and E.W. Taylor, 1969. Isolation of an actomyosin-like protein complex from slime mold plasmodium and the separation of the complex into actin- and myosin-like fractions. Biochemistry 8, 4964–75.

9. H. Ishikawa, R. Bischoff and H. Holtzer, 1969. Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. Journal of Cell Biology 43, 312–28.

10. H.E. Huxley, 1963. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. Journal of Molecular Biology 7, 281–308.

11. M. Clarke and J.A. Spudich, 1974. Biochemical and structural studies of actomyosin-like proteins from nonmuscle cells. I. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum. Journal of Molecular Biology 86, 209–22.

12. T.D. Pollard and E.D. Korn, 1973. Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. Journal of Biological Chemistry 248, 4682–90.

13. A. De Lozanne and J.A. Spudich, 1987. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236, 1086–91.

14. M.P. Sheetz and J.A. Spudich, 1983. Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature 303, 31–5.

15. R.D. Vale, T.S. Reese and M.P. Sheetz, 1985. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39–50.

16. J.A. Spudich, S.J. Kron and M.P. Sheetz, 1985. Movement of myosin-coated beads on oriented filaments reconstituted from purified actin. Nature 315, 584–6.

17. T. Yanagida, M. Nakase, K. Nishiyama and F. Oosawa, 1984. Direct observation of motion of single F-actin filaments in the presence of myosin. Nature 307, 58–60.

18. J. Kron and J.A. Spudich, 1986. Fluorescent actin filaments move on myosin fixed to a glass surface. Proceedings of the National Academy of Sciences of the United States of America 83, 6272–6.

19. Y.Y. Toyoshima, S.J. Kron, E.M. McNally, K.R. Niebling, C. Toyoshima and J.A. Spudich, 1987. Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature 328, 536–9.

20. R.W. Lymn and E.W. Taylor, 1971. Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10, 4617–24.

21. I. Rayment, W.R. Rypniewski, K. Schmidt-Bäse, R. Smith, D.R. Tomchick, M.M. Benning, D.A. Winkelmann, G. Wesenberg and H.M. Holden, 1993. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261, 50–58.

22. J.T. Finer, R.M. Simmons and J.A. Spudich, 1994. Single myosin molecule mechanics: Piconewton forces and nanometre steps. Nature 368, 113–19.

CHAPTER 27

RNA Processing: A Postdoc in a Great Laboratory

Sidney Altman

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT

The author is Sterling Professor of Molecular, Cellular and Developmental Biology at Yale University and, in 1989, shared the Nobel Prize in Chemistry with Tom Cech for the discovery of catalytic RNA. This essay is based on a talk presented at the reunion of the MRC-LMB held in Cambridge on 26 April 2003, and subsequently adapted for and published in Genetics. Naturally, some of the local elements of that talk have been modified and a discussion of topics and personalities has been added as extracts.

In 1963, Sydney Brenner wrote a letter to Max Perutz about the future of research at the MRC-LMB. He said: ‘The entry of large numbers of Americans and other biochemists into the field will ensure that all the chemical details of replication and transcription will be elucidated’ (cited by Brenner, 1988). He then went on to describe the new plans for developmental biology he was exploring. I was one of the North Americans to come and work out the details Sydney had mentioned in his letter. I came with a rather modest reputation in biochemistry. Along my route as a postdoctoral fellow at the MRC-LMB, there were moments of great luck and disappointment. At every moment, however, I felt so extremely privileged to be an associate of the greatest laboratory of molecular biology at that time. That experience, in itself, made my stay worthwhile, even if nothing of significance in science emerged from my activities.

As a child, I became interested in the great age of physics in the twentieth century. I read a lot about Einstein and Bohr with his amazingly productive group. It was too much to ask for, but I hoped that one day I might be part of such a group, particularly one like Bohr’s. Ultimately, I did join such an amazing collection of personalities, but they were in molecular biology and were located in Cambridge at the MRC-LMB.

The atmosphere of the great physics groups and their intimate collaboration and communication can be briefly reviewed in A. Pais’s books about Einstein and Bohr (Pais 1982, 1991). Einstein had finished his major efforts and worked more or less by himself, even while in Berlin, although he was an astute and responsive listener. Bohr worked constantly with several young people around him, communicating and discussing new ideas throughout the days and weeks. Several of his associates went on to great fame.

My first contact with the group, if contact is the right word, came when browsing through an issue of Nature in 1961. I saw the Brenner and Francis Crick article about the nature of the genetic code (Crick et al, 1961). I copied that article and read it. My fascination with that paper was in how anyone had been able to learn what the article said had been learned.

At that time, late in the spring of 1962, I was in Colorado, working and going to summer school as a physicist. Later that summer, I met George Gamow at a party for students. He was obviously the most colourful person there. Gamow was well known to anybody who studied physics, as I had up to that point. He made the first calculations on alpha particle decay and went on to a plethora of important contributions in physics, among them the theory of the Big Bang. He also played a critical role in the early days of the genetic code. After reading about the structure of DNA, Gamow recognised that there might be a relationship between the number of bases read in the linear sequence, three at a time in this case, and the order of amino acid residues in a protein (no discussion yet of mRNA) to make a protein. He made a simple calculation of the number of possibilities of amino acids you could code from a sequence of DNA (using the wrong number of amino acid residues) and predicted what kind of sequence you would have in a protein (Gamow, 1954). These calculations were incorrect partly because of the unknown nature of the code (overlapping, non-overlapping, etc.) and the incorrect number of amino acid residues used by nature. Nevertheless, the direction of the ideas was clear and accurate and spawned several years of intense speculation and experimentation about the nature of the code.

At Gamow’s suggestion, because of my partial understanding and an interest in molecular biology, I contacted the Department of Biophysics at the Medical School in Denver. Ted Puck was the chair of that small department. When I drove down from Boulder, I visited Leonard Lerman (Figure 27.1). That was the end of my career as a physicist. Leonard had just returned from a sabbatical year at the MRC-LMB. He and I hit it off, and I became his student. We agreed, shortly thereafter, on a topic for a thesis. In the meantime, Leonard told me about his work on acridine-DNA complexes, which he had discovered, and his life at the MRC-LMB. A few years later, in Nashville, Tennessee, where Leonard had moved and I finished my thesis, I met Sydney Brenner.

Figure 27.1. Leonard S. Lerman.

Sydney gave one of the greatest lectures I have heard in science. There were no slides. It was all about the genetic code and nonsense codons. Later, I told Sydney that I would like to work at MRC-LMB, but he said there was no space available at the moment. Disappointed, I went on, very fortunately, to Matt Meselson’s lab, and there I saw Sydney again after another year or two. He was visiting the Boston area. He asked me if I was still interested in the MRC-LMB, and when I said ‘Yes,’ he indicated that there was newly available space in the lab and suggested I might want to work on the three-dimensional configuration of tRNA, probably by NMR. Any physicist, or student of physics, could operate a big NMR machine.

I arrived in Cambridge in October 1969, just after the annual lab meeting, an important occasion that I happened to miss twice during my time in Cambridge. Very soon I had a meeting with Sydney and Francis. They told me that tRNA had been recently crystallised and that I should come by with a new, i.e. my own, plan for study. Two weeks, they said, and we will meet again. They did suggest some possible fluorometric studies of tRNA in solution.

What an idea! Come up with my own research plan! I looked around, read some uninteresting papers, and then went back to Sydney and Francis. I suggested that I look for acridine-induced mutants of tRNAs. Since they had recently completed a classic study of substitution mutants of tRNAs, the idea I presented was met with what I perceived as an attitude of great boredom. Nevertheless, they said, if that is what you want to do – go ahead. I distinctly felt that if they spoke to me again, it would be an accident. The conclusion I drew was that studies of tRNA mutants were finished.

Brenner and Smith and their junior colleagues had recently published papers on substitution mutants of tRNATyrSu3+ that demonstrated the expected change in the anticodon of the tRNA as it became an amber suppressor and then how the properties of suppression changed as other nucleotides in the sequence were altered (Abelson et al, 1970; Smith et al, 1970).

I did have a few experiments left over from my previous work with Matt Meselson, which I decided to complete. Let us remember that they concerned a DNA endonuclease, so I had some experience dealing with an enzyme that cut nucleic acids. I also gave a seminar on that work, which did not excite me very much. To my surprise, Fred Sanger was the only senior member of the lab who attended, with only a few in the audience. It took me a while to figure out why Fred was there (to hear about new DNA endonucleases) and the reasons for the questions he asked. He was working on sequencing DNA and looking for new enzymes to recapitulate the work on sequencing of RNA. Jon King, another inventive T4 person with whom I did some very slow work on T4 encapsulisation, was also there.

I did get started on making acridine-induced mutants of tRNA. John D. Smith (Figure 27.2), whom I had not previously met nor even heard much about, helped me set up the genetic system, and I worked on that for my first year. In fact, I started out with su+am mutants of tRNATyr and did succeed in making various non-suppressing mutants. These were made in Escherichia coli lysogenised for φ80su3+: the cells were induced, and the tRNA genes transferred to the φ80 itself and then examined more closely. Indeed, I had several mutants that made no tRNA, but some did revert to the Sup+ phenotype at frequencies of about a few per cent. By this time, May 1970, I was getting ready to leave the lab and look for jobs in other fields.

Figure 27.2. John D. Smith (courtesy of the MRC-LMB).

I should also say that I rented a sixteenth-century, very small, thatched-roof cottage in Barley, a few miles from Royston (Figure 27.3). It had been the priest’s cottage for many years and is on a map of the village dated 1545. My life there was very quiet and gave me a good chance to think about what was happening in the lab. It was made tolerable by my friendship with Jack Wilkerson and his family, Jack being one of the squires of the village and also a very good farmer, an amateur archaeologist and an historian.

Figure 27.3. My cottage in Barley.

I had a one-year fellowship that was rapidly coming to a close. It was a bad time for jobs: the market in molecular biology had not yet expanded, and my search came up empty. That was a time of deep disappointment.

Sometime in May 1970, Sydney suggested to me that I had better get something published or my meagre talents would be lost to science. He had one problem, not yet solved, concerning ochre-suppressing mutants of tRNATyr. The idea was to make an ochre-suppressing tRNA mutant and sequence it to show that it had the expected anticodon. Amber-suppressing mutants had already been characterised in earlier publications by Brenner, Smith and colleagues (Figure 27.4). I started the project Sydney suggested rather quickly in my spare time. Within a week or so, I had isolated the mutants, isolated the tRNAs, and started the sequencing to show that they were the correct tRNAs. This work went almost too smoothly; there were no problems. Others in the lab had previously tried this project without success. I did everything the same way they did, except that I used a new mutagen, hydroxylamine. It was the favourite mutagen of Bill McClain, another American. I had thought it was the perfect mutagen of choice from a chemical point of view, and he had a bottle full of it, ready to use. So now I had one publication.

Figure 27.4. A description of tyrosine tRNA with locations of mutations of the amber and ochre anticodons at position 35.

It is worth mentioning that I had read about Sanger’s two-dimensional fingerprinting of RNA before I came to the lab. I watched people do it at MRC-LMB, but I still felt it was an impossible method; it seemed that too much voodoo was involved. So when I had to start sequencing, I asked for some help from another American, Hugh Robertson, and soon learned that there was no voodoo, although it had seemed that way to an observer. The method worked brilliantly over and over again. I tried to emulate Bart Barrell, who could do about seven or eight fingerprints simultaneously, but I never quite succeeded. The most I could do was six.

A few weeks before my fellowship terminated, I decided to do one more experiment on the tRNA acridine-induced mutants I was making. I thought that the mutants I had that reverted at high frequencies were probably partial duplication mutants and that their finished product was too ‘damaged’ to survive for very long in the cell. Consequently, I tried to see if I could isolate a precursor tRNA molecule in the fastest way possible. My decision was influenced by the fact that every time somebody reported such an experiment in the literature, there was always a centrifugation step to spin cells down. Even a 30-second centrifugation would be sufficient, in my mind, to allow the cells to destroy any unwanted transcript. I avoided such a step and added phenol directly to the cultures. The first experiment proved that I was on the right track, as I found some large molecular weight bands running above tRNA in the gel. Their size was appropriate for partial duplications (Figure 27.5). Soon I also included one of the older base substitution mutants and noticed that it, too, had a higher molecular weight band. My colleagues, namely Bill McClain and Tony Cashmore, encouraged me to fingerprint these products (Altman, 1971). There was some skirmishing, because they wanted to see the fingerprint the moment it was developed. I think I managed to avoid that, but Tony, especially, was very excited when the fingerprint showed that I had precursor molecules.

Figure 27.5. Analysis of mature and precursor tRNAs in a polyacrylamide gel. Bands labelled X and Y are precursor tRNAs.

My fellowship ended. What was I supposed to do? Francis, Sydney and John Smith were away, but at least Sydney was accessible by mail. I wrote to Sydney explaining what I had done and asked for permission to stay, without salary, so I could work on this experiment and get the results I needed to allow me to apply for a job. Very fortunately, Sydney answered and indicated that there might be a standard, local MRC postdoc salary for one year. I am still grateful for this unexpected and generous hospitality.

The subsequent year found me identifying the processing enzyme that cleaved the 5′ sequence from precursor tRNAs (Figure 27.6). Hugh Robertson, who had extensive experience with RNases, guided me and supplied crude extracts ready to use. John Smith returned to the lab, and we worked together on mutants and other aspects of the problem.

Figure 27.6. The secondary structure of a precursor to tyrosine tRNA. The arrow between positions –1 and +1 is where RNase P cleaves this substrate. The amber anticodon is indicated at position 35, and other mutants and modified bases are also shown.

John Smith was a venerable nucleic acids biochemist. He had worked on the nucleic acids of viruses long before coming to the MRC-LMB and was an expert on identification and characterisation of nucleotides, much of it done on unlabelled material, detected by observing chromatographs under UV light. He was a quiet person but very lively in conversations about science.

We found that many previous substitution mutants of tRNAs had precursor molecules that were not cleaved properly by RNase P, our new enzyme. In addition, Bill McClain applied our knowledge to that of T4 tRNAs and, over the next few years, painted an extremely detailed picture of the genetics and biochemistry of tRNA synthesis by T4. When I went back to Yale, I followed Arthur Kornberg’s dictum that every graduate student ought to purify an enzyme before he gets a PhD and put a graduate student, Ben Stark, on purifying this enzyme. In the end, my MRC-LMB journey helped start the field of the enzymology of tRNA processing.

I did not realise what I had done with the precursor tRNAs. I knew it would get me a job, and it did. Sometime later that year, Gobind Khorana came through the MRC-LMB and was steered to me. It was hard for me to believe that this intellectual giant would want to hear about my work. But, of course, he was working on making a synthetic tRNATyr gene, and he wanted everything, including the gene sequence, characterised. He went away somewhat glum, but within a year or so he succeeded in doing what he wanted.

Paul Berg also visited the lab and wanted to find out more about my work because Dale Kaiser’s student, Paul Schedl, was working on a similar project. As I recall, nobody gave him much time, but one morning he came to me and I agreed to send him my phage strains. In fact, Schedl’s work became quite interesting as he had unwittingly isolated a temperature-sensitive mutant in the protein subunit of RNase P.

I should mention that no one had isolated a radioactive, pure precursor tRNA prior to this time. Burdon and Darnell separately had looked at putative collections of mixtures of tRNA precursors in mammalian cells and had shown that crude enzymology was involved in changing their size from somewhat larger than tRNAs to that of normal tRNAs (Bernhardt and Darnell, 1969; Burdon, 1971). That was the extent of similar work at the time. Our work started the processing and detailed enzymology of E. coli tRNA. Very soon, 30S ribosomal RNA precursors were isolated in E. coli by Apirion and Schlesinger, their colleagues, and others, and it was rapidly shown that RNase III was involved in their processing. tRNA processing was also pursued in human cells by my lab, and rRNA processing in eukaryotes had several converts. The processing of mRNA also followed in quick succession (Brookhaven Symposium 1974).

In the summer of 1970, I learned that my father had leukaemia. I went home to Montreal in the early autumn for about two to three weeks to be with my parents. My father was originally a penniless immigrant to Canada. He died while I was in Montreal. When I arrived back in the lab, again having missed the annual meeting, Sydney saw me as I climbed the stairs to the second floor and, as might be expected, asked why I had taken so much vacation. I still do not know if he was joking.

Later that autumn, Sydney came into the then large lab room on the second floor with a blue airmail letter in his hand. ‘Who wants a job at Yale?’ was the greeting, shouted loudly enough so that everyone heard him. It is my recollection that my hand shot up: ‘I do.’ Ultimately, I did receive an offer from Clem Markert, the chair of the Yale department. Of course, Markert offered me a salary that was much lower than that offered by other places, and when I timidly commented on this, his typical answer was: ‘You can be bought?’ Nevertheless, he gave me a little more money, and I took the job. (That was my first experience with Markert, an outstanding developmental biologist and a superb, courageous person who fought with the Lincoln Brigade in Spain and was a determined and brave opponent of Senator Joseph McCarthy.)

The enzymology we did on RNase P at the MRC-LMB was a solid beginning to the next phase that was carried forward at Yale: the complete purification and characterisation of the enzyme, which had, as it turned out, a large catalytic RNA subunit. It is worth remembering that in those days we were still in the millicurie era. Each experiment I did to label tRNA (or precursors) or to prepare substrates for RNase P involved putting about 3 mCi in a bubbler tube for several minutes and then extracting the labelled RNA. One day in the summer of 1971, shortly before I left, I was doing an experiment with 5 mCi in an attempt to label the wild-type tRNATyr precursor and to determine its fingerprint. In this case, without my knowledge the bubbler tube I was using developed a small hole in the water bath. A stream of highly radioactive 32P was scattered all over my bench and the surrounding area when I removed the bubbler tube.

John Smith, our radioactive safety officer, was away, and Sydney muttered, ‘I’m too busy. Go and see Fred [Sanger].’ Fred listened to me and came downstairs to the second floor. Without a word, he put on some rubber dishwashing gloves, grabbed a can of Ajax and some sponges, and knelt down to try to see how much radioactivity he could get off the floor. Not much. I objected to what he was doing because I thought I should be doing that job. Quite soon, he said that I had done enough and decided to clear the area. I stripped my clothing off, donned a lab coat, and drove home where I left the clothes in the coal shed to ‘cool’ off. Hugh Robertson called the contaminated space Yucca Flats, and some time later that whole floor and bench area were removed.

I cannot imagine a person of Fred’s reputation taking on such a modest and thankless task and refusing any help. The image of him, on his knees on the floor, trying to wash away the radioactivity I had spilled, is still fresh in my mind. There are many ways, I suppose, of exhibiting humility and greatness, and this was one of them.

I have described my history as a postdoctoral fellow at the MRC-LMB. The lab itself was such an amazing place in which to work and deserves significant comment. Mike Fuller ran the stockroom both efficiently and in a friendly fashion to ensure that we always had what we needed. The library, with a modest collection of journals, was always available and supplied with photocopies of articles on demand. There was, importantly, a collection of brilliant younger colleagues, each with his own bizarre identity. The air was bubbling with notions of both useful, practical ideas on how to go about things and theoretical notions that were interesting to discuss. The postdocs were international and vibrant. John Smith liberally dispensed his fund of knowledge, the more liberal the better you knew him. The generated ideas whizzed around the lab, many not useful, but when one was, it was recognised as such and shone brilliantly. There were also many extraordinary social and sporting functions with the postdocs and staff. I can instantly create a picture in my mind of playing football (soccer) for the MRC-LMB B team on a local village’s meadow on a Sunday morning. Needless to say, while the events I have described are mostly joyful, there were moments of great disappointment during that first year, and episodes of quarrel and disagreement among the many prima donnas in the lab.

While I was at the MRC-LMB, Sydney was steadfastly plugging away on the histology of the nematode and beginning to develop a system for mutant isolation. Occasionally, I would meet with him to discuss some science; those meetings were filled with dazzling fireworks. There were rockets and flares that fizzled and burned out, and others that cast their illuminated glow on the landscape and burned brilliantly forever. His fund of knowledge was enormous, and the ideas flowed freely. Many were not practical, but some always were.

Francis was preoccupied with chromosome structure. In fact, he surprised me on a solitary Saturday afternoon when he approached me in the hall, very excitedly, and wanted to tell me about a new theory. I was, of course, flattered but dumbfounded. I understood very little about the chromosome and the theory we were discussing, and this conversation did not further my knowledge much. Fred Sanger was working on the sequencing of DNA, I believe having already given up on using the various enzymes that he had employed with proteins and RNA (see Stretton, 2002). Max Perutz was heavily involved in trying to understand the mechanism of haemoglobin action; Aaron Klug was finishing up further details of tRNA crystal structures and getting started on nucleosomes; and César Milstein was busy with antibodies (Figure 27.7).

Figure 27.7. Several of the senior leaders in the MRC lab around 1967. Seated from left to right: Hugh Huxley, Max Perutz, Fred Sanger, Sydney Brenner; standing left to right: John Kendrew, Francis Crick (courtesy of the MRC-LMB).

Sydney still had an abiding interest in tRNA suppressors, so in the summer of 1971, Larry Soll, who had recently received a PhD from Stanford, came to give a talk on su7+. Sydney showed up after a while dressed in his usual James Dean outfit of chinos and a T-shirt with a cigarette pack stuffed inside one of the sleeves. He managed to ask some good questions and, afterwards in the hall, Larry asked, ‘Who was that far-out freak with the good head?’ That created a round of hysterical laughter from the surrounding people. I am not sure we have heard a better definition of Sydney. In any case, John Sulston, in The Common Thread, writes about Sydney as a ‘complex and powerful personality’ (Sulston and Ferry, 2002). For those of us who know Sydney, that can be considered only the essence of a very vague description. More recently, Bargmann and Hodgkin described Sydney as ‘outrageously brilliant, charismatic and witty’, a much more satisfying description when complementing Sulston’s words (Bargmann and Hodgkin, 2002).

One has to remember that Sydney and Francis shared an office, which was unheard of in the United States. That fact immediately marked the lab as unusual and interesting. Sydney and Francis needed to talk to each other. We can only imagine what wonderful conversations they had. And if one of them was not around, then whoever was in the coffee room became the focus of talks.

Conversation was always a pleasure. I enjoyed tea, mornings and afternoons, and lunch every day where everything, from local British politics to matters of serious scientific interest, was discussed.

Of course, the primary and most important feature for those of us who were new in the lab was that everybody was heard. Holding a position at the lab meant, as the senior people showed every day, that you had a respectable opinion on science and that your opinion was treated the same as everybody else’s. The dedication to science was paramount. Everybody paid attention. However, if what you said was incorrect or foolish, you were promptly told so. You were also expected to work, more or less alone, with no immediate help from senior people. The assumption was that everybody could do experiments well. The senior people taught by example: everybody was in the lab, working. These permanent, abiding lessons of how to do science have remained with me and, I hope, have been passed on to my students over the years.

I spent the last several months in the MRC-LMB trying to finish up my experiments. Two years had seemed like a lifetime. I had some reasonable results to work on, and I seemed to be able to convince people that the results were important.

My time at the MRC-LMB frequently seems to me to be the beginning and the end of my supposedly fabulous career as a scientist. After I left, except for a brief visit to finish a manuscript that Hugh Robertson and I were working on, I stayed away for many years because I did not want the MRC-LMB to guide my life. My scientific work gradually drifted away from tRNA mutants and focused on RNase P, which I still work on. Later, when I did come back for a few days from time to time, it was always a great pleasure to see Sydney and the others. Gradually, as Sydney and Francis left the lab, my friendship with Aaron Klug grew, and we have worked on RNA proteins jointly.

Work on RNase P is usually a three-to-four-year period of boredom and fright. Now we are in one of the fifth years. My lab is currently concerned with RNase P in both E. coli and human HeLa cells. In E. coli, we have identified small, intergenic RNAs that are substrates for RNase P, an indication that this enzyme is involved in determining gene expression in operons. In HeLa cells, we have shown that there are at least ten protein sub-units of the enzyme as well as an RNA subunit. We have also shown that in transient transfection experiments, the inhibition of the expression of the protein subunits does affect some but not all the subunits if one of them is specifically targeted.

There is no doubt that Sydney and the beautiful atmosphere of the lab for doing science were my greatest inspirations. Sydney, although from some viewpoints distant, arrogant or hard to deal with, became for me a very familiar person. The community of immigrants in South Africa in which he grew up was not terribly different from the immigrant community where I grew up in Montreal. He is a tremendous comic when he wants to be, as well as arrogant and deprecatory, with humour, when he feels that it is appropriate. His knowledge of biology is encyclopaedic. I learned, as a postdoc, not to speak to him unless I had something more or less correct to say. In more recent years, I have forgotten that discipline: questions can be asked that are not totally appropriate. In fact, about two years ago at Yale, Sydney suggested that what I was saying about a topic he was working on was ‘all tryptophan’, a familiar phrase to those of us who know him and probably as close as he gets to profanity in public.

Memory can be elusive. Perhaps we may consider this essay as ‘all tryptophan’ or, if that is not adequate or fitting, please be kind in selecting your own appropriate word.

Recently, John Heuser wrote in Science of the words that Sir Bernard Katz used in describing his own career. Among these were ‘a good mentor and a great deal of luck’ (Heuser, 2003). My good fortune in the first category starts with Lee Grodzins in physics at MIT, went on to Leonard Lerman, a friend and thesis supervisor, Matt Meselson, and finally, as written here, Sydney Brenner and the amazing collection of senior personalities at the MRC-LMB. In fact, very shortly after I arrived at the MRC-LMB, I saw Sir Lawrence Bragg, who came to the lab to see a model for the structure of tRNA. The names that were to become famous were all about us every day.

All the postdocs worked very hard, knowing full well that when a word of advice was needed, it was always there, down the hall, at a door that was always open.

REFERENCES

Abelson, J.N., M. L. Gefter, L. Barnett, A. Landy, R. L. Russell et al, 1970. Mutant tyrosine transfer ribonucleic acids. Journal of Molecular Biology 47, 15–28.

Altman, S., 1971. Isolation of tyrosine tRNA precursor molecules. Nature New Biology 229, 19–21.

Bargmann, C. and J. Hodgkin, 2002. Accolade for elegans. Cell 111, 759–62.

Bernhardt, D. and J.E. Darnell, 1969. tRNA synthesis in HeLa cells. Journal of Molecular Biology 42, 43–56.

Brenner, S., 1988. Foreword, pp. ix–xiii in The Nematode Caenorhabditis elegans, edited by W. Wood and the community of C. elegans researchers. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Brookhaven Symposium, 1974. Processing of RNA, vol. 26. Brookhaven Symposium in Biology, Brookhaven, NY.

Burdon, R.H., 1971. Ribonucleic acid maturation in animal cells. Progress in Nucleic Acid Research and Molecular Biology 11, 33–79.

Crick, F.H.C., L. Barnett, S. Brenner and R. J. Watts-Tobin, 1961. General nature of the genetic code for proteins. Nature 192, 1227–32.

Gamow, G., 1954. Possible mathematical relation between deoxyribonucleic acid and proteins. Dan. Biol. Medd. 22, 1–13.

Heuser, J., 2003. My little spontaneous blips. Science 300, 1248.

Pais, A., 1982. Subtle Is the Lord. Oxford University Press, New York.

Pais, A., 1991. Niels Bohr’s Times. Oxford University Press, New York.

Smith, J.D., L. Barnett, S. Brenner and R. L. Russell, 1970. More mutant tyrosine transfer ribonucleic acids. Journal of Molecular Biology 50, 1–14.

Stretton, A.O.W., 2002. The first sequence: Fred Sanger and insulin. Genetics 162, 527–32.

Sulston, J. and G. Ferry, 2002. The Common Thread. Bantam, London/New York.

CHAPTER 28

Actin Odyssey – Structure and function of actin filaments (1970–72)

Takeyuki Wakabayashi

Department of Biosciences, Teikyo University; Department of Physics, University of Tokyo

Figure 28.1. Takeyuki Wakabayashi.

It is my great pleasure and honour to be given this opportunity of celebrating the 50th anniversary of the opening of the MRC Laboratory of Molecular Biology, Cambridge.

Since I visited Cambridge in 1970, I have been working on the structure and function of actin-containing filaments. Actin filaments are a hallmark put on all eukaryotic cells and are essential for cytokinesis, cell signalling and intracellular trafficking. Many actin filaments in normal cells are unstable and their dynamic polymerisation and depolymerisation are essential for cell migration, but they can also form stable structures in muscle cells. With myosin they generate force.

From 1965 to 1970, I was studying the regulatory mechanism of muscle contraction by characterising troponin, as a graduate student of the Ebashi lab. Troponin is a calcium-binding protein indispensable for the regulation of striated muscle contraction. During determination of the molecular weight of troponin by light scattering, I found that troponin is monomeric with Ca2+ but becomes oligomeric without it.1 Meanwhile, inspired by the Hanson-Lowy model of actin filament, I put tropomyosin into two grooves of the actin double helix and added one troponin to one tropomyosin to complete the model of actin-tropomyosin-troponin.2 However, I could not correlate the calcium-induced changes of troponin with the model I proposed.

It was fortunate that Professor Alan Hodgkin came to Japan on behalf of the Royal Society to start the exchange scheme between the UK and Japan, because I was looking for a postdoc position abroad in a different discipline. He gave a talk on the behaviour of nerve membrane observed by aequorin, which fluoresces on binding calcium. It was very interesting that the calcium entry into cytoplasm could be measured directly, and I asked a question about the possibility of potassium channels being used to carry calcium ions, because the integrated time course of calcium entry appeared similar to that of potassium conductance. He responded that this was a new question and that he would think about it. A few months later, I was informed that the Royal Society had awarded me a fellowship. I was very grateful to him. After I started to work in the LMB, I had an opportunity to thank him in person, when Professor Ebashi, my mentor in Tokyo, visited his laboratory in Downing Street. It was very nice to talk to such a great and kind person. He immediately understood why the image cannot be reconstructed from the recorded optical diffraction pattern, and explained it to his colleague who joined us later.

In October 1970, I began to work at the LMB as an exchange fellow of the Royal Society and stayed until December 1972 as a short-term staff member. It was a wonderful time. People were kind and helpful. Hugh Huxley, my first mentor in Cambridge, taught me electron microscopy. Jim Spudich showed me what he had done on actin filament structure, and how to perform the image reconstruction. Linda Amos patiently taught me the details of computer programming commands. Aaron Klug, my second mentor in Cambridge, encouraged me by saying: ‘Use an armoury of methods available. Take a risk,’ and was always ready to answer any questions. When John Finch explained matters, one could feel the Fourier space as naturally as the real one. Alan Weeds and John Kendrick-Jones introduced the biochemical facilities. People working in the store managed by Mike Fuller were friendly, and those who took care of glassware were cooperative. After returning to Tokyo, I revisited the LMB and made the acquaintance of Richard Henderson, Murray Stewart and Kiyoshi Nagai. They all stimulated my mind.

I worked mainly on the ground floor to prepare proteins for electron microscopy. In the same room Nigel Unwin was working on TMV and gave good advice on negative staining and image analysis. The computational work was done on the first floor. My roommates were Ruth Sperling working on a mutant TMV and John Haselgrove working on fibre diffraction. Jan Mellema taught me how to use the Phillips microscope.

The canteen provided an invaluable forum. Due to biochemical experiments, I tended to be late for lunch. On one such occasion, Francis Crick came and kindly joined me. I was excited. He asked what I was doing. After listening to my reply, he looked thoughtful and said: ‘The regulatory mechanism is more difficult to study.’ Since then, I remember his words from time to time. The canteen did not serve supper. In the autumn of 1972, Mark Bretscher and Roger Kornberg were single. Richard Linck and I were temporarily single, since our spouses had returned to their homelands. We had supper in Addenbrooke’s Hospital, talked with enthusiasm, and then went back to the evening session of experiments. Once I asked Roger to show me how to perform slab-gel electrophoresis, which was a new technique. He knelt in front of a bench to set up carefully, so I followed. We must have appeared deep in prayer.

Before I travelled to Cambridge, Hugh Huxley wrote me a letter, in which he suggested I follow Jim’s work by showing the calcium-induced changes of filaments containing actin, tropomyosin and troponin. I agreed. I was more than happy, because I had been thinking that the functions of troponin would be better studied in the complex with actin and tropomyosin. Since I had several months before joining Hugh, I tried to follow the computational process of image analysis. Following the paper by Klug, Crick and Wyckoff, I wrote FORTRAN programs. IBM in Tokyo kindly provided the source code of a fast Fourier transform routine. The JEOL Company helped me digitise micrographs selected by a home-made optical diffractometer. The data recorded on paper tapes were processed to show actin filament in 3D. I liked to watch actin drawn on the stacked glass plates. By analysing optical diffraction patterns, the stoichiometry of actin and troponin was determined to be seven to one. Since the stoichiometry of tropomyosin and troponin is one to one, this means that one tropomyosin binds seven actin protomers.3

I settled in Churchill College and began to try to form paracrystals of actin filaments, actin-tropomyosin complex, and actin-tropomyosin-troponin complex. The former two were always able to interact with myosin, whereas the latter was inhibited unless calcium ions were added. The filaments in paracrystals were more robust against the staining procedure for electron microscopy, and the success of the whole project depended on their formation.

The first time I had occasion to remember Francis’ words was when I realised paracrystals of actin-tropomyosin-troponin complex were more difficult to prepare, especially in the absence of calcium. Ironically, this difference was encouraging, because it suggested that some changes occurred. I noticed that EGTA binds uranyl ions more strongly than calcium ions, and all other chelating reagents were the same. When the specimen with EGTA was stained with uranyl acetate, it would bind to EGTA. To avoid such complexity, the biochemical nature of troponin was exploited: when troponin was added to actin-tropomyosin after the removal of calcium-binding subunit of troponin (TnC), the system was inhibited irrespective of calcium. I was pleasantly surprised to find that Hugh had the same idea. The idea worked. The nice thing was that the paracrystal formation of such inhibited thin filaments was even more difficult!

On the occasion of the annual lab talk in 1972, I could report the azimuthal shift of tropomyosin on the actin filaments induced by troponin.4 Thanks to Ken Harvey in the photography section, the slides showing the structures in the active and inhibited states could be prepared in time. Max Perutz commented that I could have improved the presentation by putting two structures side-by-side on a single slide. I was grateful for his attentiveness to my talk. This shift explains the decrease of the second actin layer-line and increase of the third one of the X-ray diffraction pattern upon relaxation. On the basis of these changes and modelling, the steric block hypothesis had been put forward by Hugh, John Haselgrove, Parry and Squire. This hypothesis proposed that the actin-myosin interaction is blocked sterically by shifted tropomyosin. However, a crucial question remained to be answered: whether shifted tropomyosin actually covers the myosin-binding site(s) on actin.

After returning to Tokyo, I began to wrestle with this problem in 1973. It took us twelve years to answer this crucial question, and the answer was affirmative as described in the next paragraph.5 Much later, in 2001, totally unexpected features of the actin-tropomyosin-troponin complex emerged. Briefly, the extent of the tropomyosin shift in the absence of calcium differed according to the region along a molecule of tropomyosin. In the carboxyl-terminal one-third it was larger, as revealed by single-particle analysis of cryo-electron micrographs.6 A protrusion, called an arm, emerged from the main body of troponin in the absence of calcium. The arm was identified as a carboxyl-terminal region of troponin-I (inhibitory subunit) by NMR spectroscopy, with this region being electrostatically complementary to the actin surface, and the hot spot of mutations that induce cardiomyopathy.7 One of two α-helices of tropomyosin was bent to provide a single troponin-binding site, and in the amino-terminal region the coiled coil was splayed apart to form a universal joint.8

In 1973, I was offered a job at the Department of Physics at the University of Tokyo, and started to work on the determination of the myosin-binding site(s) on actin to answer the crucial question described above. The simple and straightforward idea was to compare the actin-tropomyosin-myosin complex and actin-tropomyosin. I needed a good densitometer, which was expensive. Therefore, I built one by combining a Nikon projector, a light scattering photometer I used as a graduate student, and a stage driven by two pulse motors controlled by a mini-computer. It was first used successfully to average F1-ATPase images to show that it has six major subunits and one minor one.

As a myosin moiety, the chymotryptic fragment of myosin (myosin S1 or myosin head) was chosen, because it was most homogeneous. The better images were collected with a low-dose method, and the reconstructed image of actin-tropomyosin-myosin S1 showed a complicated multi-domain structure. As a control, a better structure of actin filament was reconstructed. To objectively assign the actin moiety in the complex, the cross correlation between the actin filament and the complex was calculated as a function of rotation angles, shift distances and polarity of filament. The cross-correlation function gave a unique answer and the interface between actin and myosin could be determined unambiguously.5

Thus the myosin-binding site on actin was established: one myosin head bound to one actin protomer on one strand of the actin double helix.5 Deducing from the arrowhead appearance and the triangular shape of actin, an apex of actin was shown to point towards the barbed end. The remaining task was to show the precise location(s) of tropomyosin on actin. Twenty-eight years after I returned from the LMB, this was eventually achieved by applying single-particle analysis to cryo-electron micrographs of actin-tropomyosin-troponin: the myosin-binding site was indeed covered by tropomyosin only in the absence of calcium.6

As an extension of the image analysis of actin-myosin complex, the effect of ATP on myosin was studied. In actin-myosin S1 complex, we could show that the ATP-binding site on myosin was located on the far side from the axis of actin helix, with the actin-binding site being near the axis, i.e. on the opposite side of a myosin molecule.9 This finding prompted us to examine the effect of ATP on myosin structure itself. Electron microscopy showed that the extent of bending in the middle of the myosin head was changed by ATP. There was, however, no supporting observation by small-angle X-ray scattering. When I visited Hamburg, I met Dmitri Svergun, who said that aggregates in the sample would severely disturb his calculation. Inspired by his words, I proposed Katsuzo Wakabayashi to measure X-ray scattering of the myosin head that was purified on site at the Photon Factory at Tsukuba. The ATP-induced conformation changes were detected.10 Kazuo Sutoh confirmed the observation using FRET measurement. We collaborated to work out the positions of fluorophores so that they were correlated with crystal structures of the myosin head.11

Let me return to the actin story. The amino acid sequence of actin is as highly conserved as that of histone, the most conserved of all eukaryotes. About 90 per cent of amino acids are preserved between actins from human striated muscle and Dictyostelium amoeba. Thus, it is not surprising that Dictyostelium actin can bind tropomyosin and troponin from rabbit, and that such hybrid thin filaments can be regulated by calcium ions. Actin filaments in cells are almost always associated with tropomyosin, which is a key component of the actin cytoskeleton distributed widely in eukaryotic cells. Interestingly, only tropomyosin with its amino-terminal helices splayed apart was polymerisable, and capable of binding to the actin filament.8 The special amino acid sequence in the N-terminal region destabilises the coiled coil.

A rare exception was actin from Tetrahymena, ciliated protozoa: it cannot bind to tropomyosin. The amino acid sequence was similar, but five consecutive changes were noticed. These changes were introduced to Dictyostelium actin to generate a chimeric actin, which bound to tropomyosin poorly, as expected. This shows that residues 228–32 in an α-helix are candidates for one of the tropomyosin-binding sites. When troponin was present, even the chimeric actin became capable of binding tropomyosin. It was interesting that the hybrid actin-tropomyosin-troponin activated myosin ATPase much higher than rabbit actin in the presence of calcium ions. The first three mutations were enough to cause such higher activation.

Murray Stewart visited Tokyo and advised us that X-ray crystallography would answer the strange activation of myosin ATPase by the mutant actins. Later, before a meeting in Europe, I visited Cambridge and Murray showed me the method of crystal preparation. After the meeting, nice crystals were formed. Murray had some machine time at Daresbury synchrotron site. Because some of his crystals did not diffract well, he spared me enough time to collect actin data, which were quickly processed by Murray to visualise the first crystal structure of cytoplasmic actin (β-actin). This was a control wild-type actin. Later in Tokyo the chimeric actin was crystallised. During the summer holidays, I visited the LMB to learn data processing. At first the data collected in Japan could not be processed by a MOSFILM package. Andrew Leslie discovered the reason and kindly added a RIGAKU option. The mutated side chains, however, did not show up until a few days before I had to leave. I remember the night when the structure was determined. Unlike summer in Tokyo, it was cool outside. Admiring a starry sky, I took a short cut across a vacant car park, and walked along Hills Road to my hotel.

Max came to see the actin structure at 2.0Å resolution, which was the best resolution obtained at the time. He took his time to observe the map, and then said, ‘This is a real 2Å map.’ I explained to him that the chimeric actin poorly bound to tropomyosin and that we might be able to nail down one of the tropomyosin-binding sites. At 2.0Å resolution, more than 400 water molecules were identified. To my surprise, an ATP-binding cleft was filled with the network of hydrogen-bonded water molecules to protect ATP from hydrolysis. The adenine fitted in the hydrophobic pocket, but its five nitrogen atoms were connected with actin through hydrogen bonding. Water molecules fascinated me.12 To understand how ATP hydrolysis is activated by actin polymerisation, we generated a mutant actin that hydrolysed ATP ten times faster as if it were in the filaments. In its crystal structure, the network of water molecules in the cleft was half broken and the ATP hydrolysis cycle can proceed, because the back door to release hydrolysed phosphate was opened due to the mutation.13

Allow me briefly to come back to focus on the filament structure of actin. The inherent flexibility of filaments has been a problem for high-resolution electron microscopy. To cope with this difficulty, single-particle analysis was applied to straight stretches of filaments in the presence of phosphate, which stabilises and straightens filaments. The resultant cryo-EM map enabled us to observe some of the α-helical backbone and large side chains, and to build a quasi-atomic model of F-actin with magnesium ions and phosphate ions.13

In the 5 or 8Å resolution map (Fourier shell correlation = 0.143 or 0.5), the outer domain rotated against the inner one as compared with G-actin13. Then the DNase I binding loop interacted more easily with the proline-rich loop (residues 108–112) of the neighbouring protomer. The stacking interaction between proline-109 and histidine-161 was broken. To mimic this, proline-109 was mutated to alanine. In the crystal structure, the interaction was broken, as expected. The mutant actin showed a tenfold increase of ATPase activity. Taken together, the breakage of the stacking interaction appeared to be the key step to couple polymerisation with ATP hydrolysis. A cylindrical cavity flanked by three protomers was found. This long cavity appeared to be the only route for the hydrolysed phosphate to access the external solvent, and would play an important role in the treadmilling mechanism by delaying the phosphate release. A number of divalent cations and anions had been identified in G-actin crystals. One of the cations was shared by two protomers in F-actin to reinforce the vertical interaction and explains the effectiveness of magnesium ions to induce polymerisation. One of the anions was shared by three protomers in the central region, and the binding site appeared to be occupied by phosphate, which stabilises filaments through strengthening the ternary interaction.

I have revisited the LMB almost every three years since 1977. Once, I saw Max working. He kindly paused so I could take a photo. He was washing red blood cells, presumably for his spectroscopic experiments. It was nice to see him and he was very encouraging to me. When I was working in the LMB, I sometimes saw him after supper. He came back to the lab and refined the haemoglobin model manually until late at night. It was also encouraging for me to see Fred Sanger with a micropipette in his mouth. I was amazed at how skilled Hugh was at dissecting skeletal muscles.

My LMB-philia appears to be infectious to my graduate students. Yasuo Shirakihara worked with Phil Evans, Chikashi Toyoshima with Nigel, Masashi Suzuki with Aaron, Keiko Hirose with Linda, Yoshiyuki Matsuura with Murray. I am very grateful to the LMB, which nurtured not only me but also my graduate students. They all grew up to become outstanding structural biologists.

I hope that the new LMB will inherit the original spirit of supporting young scientists and continue to be the international centre of molecular biology.

Last but not least I would like to thank everybody, whose help made my actin odyssey possible and enjoyable. Thank you very much indeed. I thank Ken-ichi Wakabayashi, who was born in Cambridge, for reading the essay.

REFERENCES

1. Wakabayashi and Ebashi, 1968. Reversible Change in Physical State of Troponin Induced by Calcium Ion. Journal of Biochemistry 64, 731–2.

2. Masaki et al. (1969) Molecular Biology of Muscle Contraction III: Structural Proteins of Muscle, Regulatory Proteins. KAGAKU 39, no. 5, 268–9 (in Japanese).

3. Ohtsuki and Wakabayashi, 1972. Optical Diffraction Studies on the Structure of Troponin-Tropomyosin-Actin Paracrystals. Journal of Biochemistry 72, 369–77.

4. Wakabayashi, H.E. Huxley, Amos and Klug, 1975. Three-Dimensional Image Reconstruction of Actin-Tropomyosin Complex and Actin-Tropomyosin-Troponin T-Troponin I Complex. Journal of Molecular Biology 93, 477–97.

5. Toyoshima and Wakabayashi, 1985. Three-Dimensional Image Analysis of the Complex of Thin Filaments and Myosin Molecules from Skeletal Muscle. V. Assignment of Actin in the Actin-Tropomyosin-Myosin Subfragment-1 Complex. Journal of Biochemistry 97, 245–263.

6. Narita et al, 2001. Ca2+-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy. Journal of Molecular Biology 308, 241–61.

7. Murakami et al, 2005. Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin. Journal of Molecular Biology 352, 178–201.

8. Murakami, M. Stewart et al, 2008. Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T. Proceedings of the National Academy of Sciences of the United States of America 105, 7200–7205.

9. Tokunaga et al, 1987. Location of the ATPase site of myosin determined by three-dimensional electron microscopy. Nature 329, 635–8.

10. K. Wakabayashi et al, 1992. Shape Change of the Myosin Head induced by MgATP, detected by small-angle synchrotron X-ray scattering in solution. Science 258, 443–7.

11. Suzuki et al, 1998. Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps. Nature 396, 380–83.

12. Matsuura, M. Stewart, Kawamoto, Kamiya, Saeki, Yasunaga and Wakabayashi (2000) Structural Basis for the Higher Ca2+-Activation of the Regulated Actin-activated Myosin ATPase Observed with Dictyostelium/Tetrahymena Actin Chimeras. Journal of Molecular Biology 296, 579–95.

13. Murakami, Wakabayashi et al, 2010. Structural Basis for Actin Assembly, Activation of ATP Hydrolysis, and Delayed Phosphate Release. Cell 143, 275–87.

CHAPTER 29

Memories of the Lab and Cambridge, 1971–3

Henry Epstein (1944–2013)

Department of Neuroscience and Cell Biology, University of Texas, Galveston, TX

Figure 29.1. Henry Epstein at his polarising light microscope inspecting worm muscle mutants, circa 1973.

PREPARING FOR THE LMB

My introduction to the Medical Research Council (MRC) Laboratory of Molecular Biology (LMB) at Cambridge preceded my formal arrival by several years. Lubert Stryer, my mentor at Stanford’s Biochemistry Department, had been a postdoctoral fellow in John Kendrew’s group where he determined the difference Fourier synthesis between ferrimyoglobin and azide-ferrimyoglobin. While a medical student at Stanford, I worked with Lubert to study the kinetics and equilibria of ferrihaemoglobins. This study provided early evidence for the ‘breathing’ of subunits in a protein molecule influencing its interaction with a small molecule.

A highlight of this period was a visit from Max Perutz and his marvellous lecture on the allosteric interactions of haemoglobin. I met Max at a social gathering, and proudly sent him my first paper, which was published in the Journal of Molecular Biology (JMB). My subsequent JMB paper on the structural flexibility of antibodies caught the attention of César Milstein.

I had already been thinking more about research oriented to cells and organisms. My next introduction to the LMB occurred when Francis Crick visited Stanford’s Department of Biochemistry to give a ‘chalk talk’ on new work in his new Department of Cell Biology at LMB in addition to his formal lecture. At this point in time, I had already been thinking more about ‘biological’ research, and Francis’s brief description of Sydney Brenner’s innovative work on the nematode worm piqued my interests in developmental biology and neuroscience, ones that I had maintained through my undergraduate and medical school days.

Other connections with the lab came from my postdoctoral work in Christian Anfinsen’s group at the National Institutes of Health (NIH). Chris had been a visitor to Fred Sanger’s laboratory in the mid-1950s and had been a close colleague of Fred’s to whom he introduced the use of radiochemicals in protein sequencing. During this time at Cambridge, Chris also interacted with Vernon Ingram who demonstrated the amino acid substitution in sickle cell haemoglobin.

David Davies, a member of the team that produced the first structures of myoglobin and haemoglobin, was another mentor of mine at NIH who had LMB experience. In addition to teaching me the rudiments of X-ray diffraction and protein model building, David has been a long-term friend. En route to Cambridge, I met both Alan Fersht and Peter Rigby, then junior LMB staff and affiliates of Jesus College, who not only eased my introduction to Cambridge, but became long-term friends of mine as well.

All of these experiences primed me for my time at Cambridge.

THE MOST UNFORGETTABLE CHARACTER: SYDNEY BRENNER

The above appellation reflects not only Sydney’s remarkable intellect, persona and wit, but also the permanent effect Sydney’s laboratory had upon the totality of my research career.

In 1969, I sent my first letter to Francis Crick, expressing my interest in working in the Cell Biology Division of the LMB. For months, I heard nothing. I then wrote to Sydney, only to receive an obscure reply. However, my further correspondence with Sydney led to my receiving the opportunity to interview with Sydney in Cambridge in June 1970.

In Sydney and Francis’ view, I was a problematic candidate for a cell biology position. Sydney and Francis viewed me as a biophysicist. They entertained various ideas of what experiments I could do with worms; many of these experiments were accomplished by John White and colleagues several years later. In a marathon four-hour interview session, Sydney recognised that my education in biology and medicine might prove useful to basic research in cell biology. Furthermore, we both agreed that understanding the structure and function of even one gene in the worm would be of great importance to developmental biology, evolutionary studies and genetics (genomics, recombinant DNA and DNA sequencing were yet to be invented, either at the LMB or in the US).

Sydney closed our session by stating that he would need to present my candidacy to the ‘Board’. I expressed then that, in terms of knowing where I was going next, time was of the essence because I would need to acquire the fellowship funds for support. Sydney stated that I should come in first thing the next morning with a ‘good reason’ why I should be accepted. I returned the next day, providing him with a perfunctory statement on my candidacy. He handed me a letter, typed and signed the day before, notifying me of my acceptance as a postdoctoral fellow. Such was Sydney’s wry wit, par excellence.

I was also exposed to Sydney’s unforgettable intellect. Sydney is a scientific polymath, originally a cytogeneticist, then a medical student, then a physical chemistry graduate student, and finally a geneticist-biochemist-molecular biologist. Even knowing all this, I was nevertheless surprised to see the three volumes of Courant and Hilbert’s Methods of Mathematical Physics on a bookshelf in one of Sydney’s rooms at King’s College. I asked him: ‘Did you actually work through the treatise?’ He answered: ‘About a third.’ I was duly impressed because the treatise was written for mathematicians only. This is one of many examples of Sydney’s wide-ranging intellect.

Sydney’s overall persona was an even greater influence upon me and others working with him than was his wit and intellect. His professional character was such that he might be at the LMB – either directly working with or talking to one or more of us – at any time of night or day. What he transmitted was a philosophy of how to do science, including a thorough understanding of the scientific method and a disciplined approach to judging important scientific problems.

MENTORING BY FRANCIS CRICK AND FRED SANGER

Francis and Fred, although clearly iconic figures, were down-to-earth, friendly and gentle people with whom I had both instructive and pleasant conversations. Their dedication to the mentoring of young scientists was exemplified by the establishment of ‘Crick Week,’ when both junior and senior scientists presented their research, and the ‘King’s College Bull Sessions’ sponsored by Fred, where younger scientists could present more complete renditions of their ideas and accomplishments over complimentary beer. In addition to these professional events, both Francis and Fred invited me to various social events.

My talks with Francis covered crystallography, cell biology, protein folding and genetics. I recall that one of our conversations got so loud and intense that Sydney threw me out of Francis’ and his shared office.

Many American scientists believed that Fred was a highly proficient technologist, who had little interest in theory. But this was a false impression. As early as 1949, Fred realised that genes must be encoding amino acid sequences and that certain mutations produced amino acid substitutions. As Fred stated in his biographical sketch in the Annual Reviews of Biochemistry, his motivation for pursuing DNA sequencing was to find a more efficient method of determining the sequences of the proteome of organisms.

Fred was a sponge of information. While a young researcher at the University Biochemical Laboratories, one of Fred’s neighbours was Gregorio Weber, the pioneer of fluorescence polarisation in the study of protein dynamics. Fred and I had an in-depth discussion of my application of Weber’s methods to demonstrating segmental flexibility of an antibody molecule.

THE FOLDING CLUB AND MAX PERUTZ

At the time of my arrival in Cambridge, intense interest in protein folding had developed. Sydney and Francis had postulated that the problem could be solved by the synthesis of small peptides of varying sequences. The LMB hired Bob Sheppard, an organic chemist of peptide synthesis, to carry out this programme. To stimulate him and others, Max organised a ‘Folding Club’. I was invited because of my recent work on protein folding intermediates at NIH.

A group of young scientists who were all to make their mark on the folding problem participated in the Folding Club, including Cyrus Chothia, Tom Creighton, Alan Fersht, Joel Janin and Michael Levitt. The meetings were exciting not merely because of our shared interest in tackling a problem, but also because of the community of talented friends that participated in the club.

Max Perutz, when providing me with a tour of the atomic model of haemoglobin, informed me of one of the most important hard facts critical to understanding protein structure and folding: there are only three invariant residues (all in the heme pocket) in all known members of the globin family. What this meant was that multiple, diverse polypeptide sequences could fold into very similar structures.

THE MUSCLE GROUP AND HUGH HUXLEY

Hugh Huxley and his biochemical associates, John Kendrick-Jones (Jake) and Alan Weeds were extremely supportive and inclusive of me and my efforts. Because I was initiating studies on worm muscle, I requested every paper that Hugh Huxley had published on muscle. It was a steep learning curve, but a necessary one. Jake worked with me through a preparation of myosin and paramyosin from the scallop. Hugh also introduced me to electron microscopy. These lessons would serve as the foundation for my future work in C. elegans. In addition, I was included in research discussions of their Muscle Group which were an excellent introduction to thinking in the field. A special treat was the sabbatical visit of Andrew and Eva Szent-Györgyi to the Muscle Group. Over the years, Hugh, Alan, Andrew and Jake have crossed paths with me multiple times during scientific meetings, seminars and the like.

WORM MUSCLE

One of the projects Sydney suggested to me was to investigate whether any of the UNC mutants affected known muscle proteins. The raison d’être for this investigation was an analogy to Sydney’s path-breaking work with T4 bacteriophage head protein that led to the demonstration of co-linearity between genes and proteins. One must remember that in 1971, recombinant DNA was not yet invented and introns had not yet been discovered. The relationship between genes and proteins led me to request and then study every paper that Sydney had published on T4 phage.

Because the goal was to identify a gene directly related to protein, we used a very simple strategy taken from Uli Laemmli and Jonathan King’s phage work: running SDS-PAGE gels on representative alleles of unc loci which had abnormal muscle by electron microscopy or were paralysed. This strategy was based upon the preferential action of the mutagen ethyl methane sulfonate (EMS) to produce G-C to A-T transitions, which would then lead to a high frequency of stop codon mutations as in T4 phage. There was a second underlying assumption that there was only one gene for each type of protein and no functional redundancy – as outlined in Francis’ 1971 Nature article.

In fact, there is a significant family of muscle myosin heavy chain genes (four) as well as non-muscle myosins of various classes in C. elegans. So how did our strategy work? In animal cells, EMS was also known to produce deletions of various sizes. After one year of slogging through genes (complementation groups) represented by single mutations (canonical alleles), I realised that one unc complementation group at that time had multiple alleles, unc-54. The e675 allele was then studied and surprisingly, part of the myosin heavy chain band moved to higher mobility, indicative of lower size, thus identifying the unc-54 product. The smaller product was later shown to be caused by an internal, in-frame deletion, not an uncommon event for large proteins. Further work showed that the body-wall muscles and the pharyngeal muscles each expressed two myosin heavy chains – one of the first, well-established examples of a multi-gene multiprotein family.

For the next ten years, in my own laboratories at Stanford and the Baylor College of Medicine, my colleagues and I studied the molecular, developmental and cellular consequences of the myosin heavy chain multiplicity. Two significant outcomes of our work were that each myosin heavy chain isoform assembles only with an identical chain, leading to two populations of myosin homodimers in each body-wall muscle cell. The myosin homodimers then assemble differentially to form different regions of the resulting thick filaments, a totally unpredicted result, still not fully digested by members of the muscle field.

A side-project for me at LMB was the finding that the then sole allele of the unc-45 locus was temperature-sensitive (ts), the first ts found in C. elegans. Its effects on sarcomere organisation and movement appeared to be reversible during the embryonic and larval stages, becoming fixed at adulthood. I speculated that this gene specified an assembly function.

Our armamentarium of antibodies, rigorous thick filament purification and mutants from several laboratories permitted us to ultimately characterise unc-45 as a protein interacting with the body-wall myosins. Further biochemical work has shown that unc-45 is a molecular chaperone functioning to mature the myosin head into a fully allosteric protein machine. The effects of unc-45 on sarcomere assembly are primarily due to its regulation of the flow of fully native myosins to filament assembly or alternatively, non-native myosins to ubiquitination and proteasomal degradation.

This work started as a side-project in Sydney’s lab, but has now blossomed into a small field comprising at least twelve research groups. Unc-45 proteins are nearly ubiquitous in fungal and animal cells with specific isoforms for heart, skeletal muscle, and the actin cytoskeleton. This has led to findings implicating unc-45 in breast and ovarian cancer metastasis and in heart development and failure.

CODA

Although I have published on C. elegans from 1974 to 2011, my work has now taken me to fields and interests that I did not imagine in 1971. Ironically, with unc-45, I am back studying protein folding, my earlier scientific interest, but from the more biological perspective that I learned from Sydney and the LMB.

Another set of memorable experiences was my connection to Jesus College, orchestrated by Peter Rigby. In order to learn first-hand about Cambridge education, I supervised medical students in biochemistry and pharmacology. Because I did not have a ‘proper’ postgraduate degree, I was inducted into the Graduate Society as well as having High Table privileges. This led to my rowing in the ‘engine room’ of the Graduate VIII, which ‘bumped’ all four days at the May races and led to my being awarded a ceremonial oar that has been a valued possession of mine. My connections with Jesus exposed me to college culture and have led to my admiration for undergraduate education at Cambridge.

A final note is the importance of my work with two remarkable members of the LMB laboratory staff, Rita Fishpool and Nichol Thomson. My experience with them informed the manner in which I choose laboratory staff: selecting those who view such work as a professional career and not a stepping-stone to graduate or medical school. Much of my work has been with Irving Ortiz – my research colleague of 29 years – and, more recently, with Shumin Li for the last eleven years.

CHAPTER 30

Wandering Spot (1972)

Tom Maniatis

Department of Biochemistry and Molecular Biology, Columbia University Medical Center, New York, NY

and

Mark Ptashne

Memorial Sloan Kettering Cancer Center, New York, NY

Figure 30.1. Mark Ptashne.

It was 1972 and Mark Ptashne and Fred Sanger were brushing up their skiing at a winter Keystone Meeting. Ptashne had made arrangements to go to London soon thereafter, on his first ever out-of-his-own-lab experience, to study tumour viruses. But Fred, in his talk, gave the impression that DNA sequencing had gotten easy, and, perhaps not by chance, Ptashne ensnared him on a long ride up a ski lift. By the time they reached the bottom, it was decided that Ptashne would go for his sabbatical to Fred’s lab to determine the sequence of an operator of phage lambda. Tom, then a postdoc in Ptashne’s lab, had deduced that the two λ operators consist of multiple repressor binding sites, and we wanted to know the sequence of at least one of its 27 base pair elements. What Mark did not tell Fred was that he was bringing along Tom; otherwise, how would any work get done?

Mark and Tom arrived at the MRC only to find there was – as always – no extra room. Fred calmly created space – Fred would share his tiny bench with Mark (!) – which undoubtedly delayed the development of ‘Sanger’ sequencing method by several months – and Tom would share a little bench space with the American postdoctoral fellow John Donelson positioned next to a young Scottish PhD student, John Fiddes. Perpendicular to this crowd was Bart Barrell’s bench, which was large enough to accommodate autoradiograms of two-dimensional RNA fingerprints. Other notables in and around Fred’s lab were Elizabeth Blackburn, John Sedat and Ed Ziff. This was the most crowded lab we have worked in before or since, but once we were in a routine people moved around comfortably, and the atmosphere was collegial and interactive. There certainly was not room for individual desks, so all work on notebooks and manuscripts was done in the library down the hall.

Prior to 1972 DNA sequences were inferred from RNA transcripts using RNA fingerprinting methods, and no one was better at this than Bart Barrell, then a technical assistant in Sanger’s lab. However, we decided to sequence the DNA directly using a laborious method Fred had worked out a year before. This involved purifying large amounts of phage λ operator DNA and then priming DNA synthesis on this DNA using DNA polymerase I. The DNA was synthesised in the presence of Mn++ with a mixture of labelled ribo-and deoxyribonucleotide triphosphates with one or more of the four triphosphates absent. The combination of chain termination and cleavage of the DNA transcripts with specific ribo and deoxyribo nucleases made it possible to infer the DNA sequence by fractionating the nucleic acids using an archaic method called two-dimensional ‘homochromatography’. This short-lived approach became known as ‘the wandering spot’ method because the labelled oligonucleotides appeared as a trail of spots wandering up the chromatography plate. The angle and extent of wandering of oligonucleotides that differ by a single nucleotide made it possible to discern the sequence.

Considering the technical advances that followed, it is difficult to appreciate how hard it was then to sequence this short DNA fragment. Virtually none of the enzymes or reagents were commercially available, and reagent preparation was a labour-intensive and time-consuming activity. For example, Maniatis purified the restriction enzymes used, and synthesised and purified all of the 32P-labelled RNA and DNA nucleotide precursors. Ptashne, when not talking long distance on the phone (much to Fred’s consternation), spent a considerable amount of time travelling back and forth to London where he took violin lessons from Emmanuel Hurwitz. While all these time-consuming events were happening, Fred and Alan Coulson were developing the method that would form the basis of sequencing the human genome fifteen years later.

The time was brief but the memories vivid. One day Ptashne passed Crick’s office, door open, and saw a pile of Scientific American magazines on his desk. Crick was reading one of them. When asked why, Francis said: ‘When trying to learn a new field the hardest thing is to get the general idea. You can then always fill in the details.’ A lesson Ptashne won’t forget. Morning and afternoon tea – they called that stuff tea, sometimes coffee – was always the high point of the day, darkened only by the lengthening shadows that overtook Cambridge starting around 4.00 pm every day. We agreed it was a good thing that the windows did not open, or there might have been more depressed Englishmen throwing themselves out. Actually they seemed curiously cheerful – the burden fell on us Americans who were used to seeing the sun occasionally. One day matters got worse when Max Perutz, the gentle boss, roamed the building, turning off all the lights to placate the community during the 1973 oil crisis. This crisis also meant rationing heat in our freezing flats, and the petrol required for Sunday trips to London (which were often not possible to make by train because of frequent strikes). Correspondence across the Atlantic was curtailed by postal strikes. However, so long as you could digest the mystery meat at the hospital’s canteen or risk the local Indian food, you could survive. Bart’s strategy was to undergo weekly purges by spending Saturday afternoons in his favourite pub.

In the end we got the DNA sequence. We identified twofold symmetric sequences, one of which we later showed to be the recognition sequence for a repressor dimer, we located the position of an operator constitutive mutation within the symmetric sequence, and we were able to infer the position of the RNA polymerase binding site relative to the start point of RNA transcription. A direct overlap between the repressor and RNA polymerase binding site provided a key mechanistic insight into repression. As the science progressed, the big question remained unanswered: what ARE the rules of cricket? Nevertheless (or perhaps because of that), for us it was a visit of a lifetime.

CHAPTER 31

Memories of the LMB (1973–6)

Sarah Hitchcock-DeGregori

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ

Figure 31.1. Left to right: Sarah Hitchcock-DeGregori, Joyce Baldwin, Betsy Goldsmith (formerly Heidner), in Paris, December 1973.

Figure 31.2. Sarah Hitchcock-DeGregori, Tom Pollard, Dennis Bray. Gardone Riviera Meeting, 1974.

In September, 1973 I arrived in Cambridge, after a spring and summer of Watergate hearings (that led to President Nixon’s resignation), soon to be followed by the Saturday Night Massacre that found Archibald Cox (the first special prosecutor for the Watergate scandal) in Cambridge hiding at a friend’s home with the phone under the pillow (when phones were attached by wires to the wall) to avoid the press following his resignation. Needless to say, Cambridge was a world apart from the US, though at the time many of us Americans felt deprived of the daily buzz that came with opening the New York Times and being glued to the news about Watergate on television. For me the excitement of working at the LMB, making new friends and living in Cambridge soon compensated.

September, 1973 was the beginning for me of three years in Hugh Huxley’s muscle group within Structural Biology. It was an exciting period in biology. With the Central Dogma fully established, molecular biology had recently been declared ‘dead’ by some leaders in the field who moved on to be pioneers in other areas of biology. The research going on at the LMB during the 1970s led to major discoveries that changed the direction of science and medicine, and resulted in multiple awards to those there at the time, to the leaders and to others just launching their careers.

Like many, my curriculum vitae looks like a series of planned events, but it is the result of a sequence of choices, often resulting from chance events, of taking one path over another. If I had stayed on the expected path I would have started a ‘real’ job in 1973, a tenure-track faculty position in the United States after a three-year postdoc, and I even had job offers! It was my good luck that Hugh Huxley was on sabbatical in Andrew Szent-Györgyi’s lab at Brandeis University in Waltham, Massachusetts, where I was working on thin filament regulation of muscle contraction. Lots was happening in the muscle field. The Hanson-Huxley sliding filament model was in the textbooks, the details being deciphered but far from understood. Richard Lymn and Ed Taylor in Chicago had worked out the basic kinetic mechanism of myosin, now the paradigm for all cytoplasmic motors and G-proteins. Troponin was discovered by Setsuro Ebashi to be the Ca2+-regulator of skeletal muscle contraction through binding to actin and tropomyosin. The troponin components and regulatory mechanism were an active focus in several labs around the world. Andrew Szent-Györgyi and Jake Kendrick-Jones, when Jake was a postdoc at Brandeis, had found that Ca2+ regulates contraction by binding directly to myosin in many invertebrate muscles. This is now known to be how cytoplasmic myosins are regulated as well.

At the time Hugh arrived in the Szent-Györgyi lab in early 1971 to learn some biochemistry, I was the only one working on regulation in vertebrate muscle, so we worked together. I showed him how to do actin binding assays in the ultracentrifuge and run SDS gels while he taught me how to make paracrystals and holey grids for negative stain, and how to use the EM. Our collaboration at Brandeis was successful, showing the Ca2+-sensitive binding of troponin components to actin-tropomyosin, and resulted in a paper that described a conceptual model that remains valid today. The particular gels in the paper were from an experiment Hugh ran, attesting to his expertise as a biochemist.

When Hugh invited me to come to the LMB to do structural studies on the thin filament, it was an easy decision; the MRC was Mecca. Many successful molecular biologists in the United States had worked there. Since I love to travel, an added attraction was the adventure of living in another country, and Cambridge in particular. The specific project was to do three-dimensional reconstructions of the regulated actin filament (actin-tropomyosin-troponin), with and without Ca2+. Peter Moore, David DeRosier and Hugh had published the first 3D reconstruction of the actin filament, and it was the time that Hugh and John Haselgrove had postulated the steric blocking model of muscle regulation. The goal was to learn if movement of tropomyosin on the actin filament was Ca2+-dependent, as predicted by the fibre-diffraction studies of living muscle in which Ca2+-dependent changes in the second layer line led to the steric blocking model. With my experience with the regulatory proteins and their interactions, I was excited to be able to work on the project.

Jim Spudich and Taki Wakabayashi had previously worked on thin filament structure, and I continued at the same lab bench. Solutions on the shelf labelled JS and TW held my optimism for success. I shared the lab with Nigel Unwin who was developing low dose EM methods that were essential for his and Richard Henderson’s success with purple membrane protein structures, and with Murray Stewart, and later John Murray. The room also housed the Philips 300 electron microscope that meant John Finch and Linda Amos were frequent visitors. Across the hall were Alan Weeds, Jake Kendrick-Jones, their long-time assistants Brian Pope and Ross Jakes, and their students and postdocs. At the beginning my desk was in the ‘model room’, where I was dwarfed by historic models of myoglobin, haemoglobin and other structures that made the LMB famous. I’ll cut to the chase and say I was unsuccessful in determining the structures as planned for a variety of reasons, not the least being my sketchy background in physics. Three-dimensional structures to prove the model were many years ahead. I am grateful to Hugh for his understanding that I did not wish to continue on the project after more than a year with lots of help from Linda Amos and Nigel Unwin, and for giving me the freedom to do a different project that I had developed.

The mid-1970s was the time when the non-muscle motility field burst forward. Dennis Bray was working in that area, with his graduate student, Keith Burridge. While I did continue my work on muscle thin filament regulation, an exciting time for me resulted from a chance encounter at a conference with Uno Lindberg, then at the Wallenberg Laboratory in Uppsala. He had discovered that monomeric actin forms a high affinity, 1:1 complex with DNase I, the pancreatic digestive enzyme, and inhibits its activity. This meeting led to a collaboration with Uno in which we showed that DNase I depolymerises actin, the first actin depolymerisating protein. John Murray and I later tried to isolate proteins from brain that affected actin polymerisation. Miraculously, given the large number of proteins that have since been identified, we failed, but it was great fun. My work with the Lindberg lab eventually brought crystals of the actin-profilin complex to the LMB in a collaboration with Richard Henderson and then Clarence Schutt. In the years since I was at the LMB Jake Kendrick-Jones and Alan Weeds both shifted their research to the non-muscle motility fields.

In addition to having the freedom and encouragement to do unscripted experiments, it was easy. This was because of the operations staff, headed by Mike Fuller. When I would need some small piece of equipment, such as a water bath or fraction collector, I’d ask Mike and within a day one would appear on my bench. The men in the stores also seemed to have everything, and even more amazing to me, knew where to find it despite no obvious cataloguing or accounting system. They were always helpful, and loved Americans, remembering to us how our country helped Britain during the war. I recall visiting the LMB several years after I left, and Mike came to me saying, ‘I have some post for you,’ as if I had been away on holiday for a fortnight.

It’s hard to think about life at the LMB without mentioning coffee, lunch and tea, obligatory for the muscle group. More than anything else the canteen is what set life at the LMB apart from other places I have been. If I was in the middle of an experiment such that I couldn’t take a break, lab members would ask if I was feeling poorly. It was in the canteen, overseen by Gisela Perutz and run by Joy Fordham and Shirley Davis, that I made many friendships and learned about the work going on throughout the LMB. I made a point of sitting with people from different groups, not just from the muscle group and Structural Biology, but from all the divisions. The LMB at that time was small enough and, thanks to the picture gallery of everyone, it was easy to meet people. There were lots of short-term visitors too, on sabbaticals or just passing through. The canteen is where we discussed a lot of science, and where I learned about the exciting developments at the LMB. Those included the DNA sequencing of the phiX174 genome in the Sanger complemented by sequencing the bacteriophage proteins in the Hartley lab, with the ongoing competition of how many nucleotides/amino acid residues could be sequenced per day, whether the DNA or the protein was ‘ahead’; then one day the then striking discovery of overlapping genes where a given region of DNA encoded two proteins by using different reading frames. There was the tRNA structure with its unanticipated twists and turns, the deciphering of the nucleosome, the painstaking lineage analysis of C. elegans (a classical embryology experiment that led to the discovery of lineage-specific programmed cell death), Barbara Pearse’s discovery of clathrin, and discussions of the idea of what came to be monoclonal antibodies, to name a few. It was in this atmosphere that I discussed my current results with colleagues as well as ideas for future work, both short and long term. Some days I felt as if I spent more time in the canteen than in the lab!

Memories of two particular afternoons at tea have stayed with me. One was the day after a ‘Bull Session’ at King’s. These were informal evening seminars presided over by Francis Crick. A friend of mine, Len Lutter, was talking about his work on the nucleosome, trying to understand the arrangement of the DNA in it using a chemical approach. When I asked why they were assuming a symmetric arrangement, Francis boomed: ‘Of course, it has to be symmetrical!’ There might have been a ‘darling’ thrown in too. Others in the room were taken aback almost as much as I was, and there was a lot of twitter about it the next day. At tea, Francis joined me without talking about it; I think it was the closest he could come to an apology.

Then, as now, having a crystal structure of a protein was the gold standard. So, Richard Henderson, always the cheerleader, helped me set up troponin to crystallise. One Friday afternoon before tea we were excited to find crystals. Of course, we couldn’t miss tea, so we bounced up to the canteen, and then Richard set up the crystals in the diffractometer over the weekend. By then the word had spread. Hugh was hopeful there would be good crystals and that I would stay in Cambridge to work on them. On Monday, with no diffraction pattern characteristic of a protein, Richard did the ‘ping’ test with a needle. If it were protein the crystal would be soft and smush; and if it were salt it would be hard and ‘ping’ away from the needle. It pinged. At tea on Monday afternoon, Max came over to me, and sincerely said: ‘I’m so sorry.’

The atmosphere I’ve just described at the LMB was no accident; it was that purveyed by Max Perutz, then director. He interacted in his modest manner with everyone, being ‘Max’ to all. When he received his Companion of Honour (CH), after returning from Buckingham Palace he showed his medal to everyone, from scientists to those in the shop, as a way to share the honour. Even though the structure of the groups was hierarchic, and as an outsider I was at times taken aback by manifestations of social class, Max made the society of science in the LMB classless.

There was also a lot of fun outside the lab. We had dinner parties, dance parties, trips to the pub. We punted on the Cam, attended May Balls and feasts at the colleges. I was fortunate to have dining privileges at Churchill College that afforded me a view of the university outside the LMB. Living in town on St Paul’s Road, I could walk or bike throughout Cambridge. My landlady had a beautiful walled garden and gave me a little corner where I could try my hand, beginning my life long love of gardening. I especially loved the Cambridge market, the Botanical Garden, the Backs, and access to the colleges and their gardens that was possible at the time. I was also close to the train station, making it easy to go to London. I took advantage of opportunities to travel both in Britain and to the continent for meetings and holidays.

Finally, some thoughts about being a woman at the MRC in the 1970s: I had attended a college for women in the United States, and the muscle field had several prominent women. In graduate school in the US in the late 1960s almost half the students were women because so many men had been drafted during the Vietnam War. The chair of the department was the prominent physiologist Bodil Schmidt-Nielsen, daughter of the Nobelist August Krogh. For these reasons I was surprised to be one of few women at the postdoctoral level at the MRC. I may have been the second or third ever in the muscle group, such that there was a certain wariness upon my arrival. I can think of only a few other women doctoral or postdoctoral trainees at the LMB at the time, including Elizabeth Blackburn, Mary Jane Gething, Janet Mertz and Barbara Pearse. Among the permanent staff no woman served as a group leader and only a few had PhDs. There were others with first degrees who functioned at a much higher level, some of whom went on to complete their doctorates and become established in their own right; Linda Amos, Joyce Baldwin and Daniela Rhodes in Structural Biology come to mind. There was no particular encouragement for women and there were no role models. At times, quite the opposite. One evening Linda Amos and I were in the lab, as was often the case for postdocs and graduate students. A man came in, and finding us, asked what we were doing there at night, as if we really should be home, Linda in particular since she was married. But times were changing. As I was preparing to return to the United States Alan Weeds told me that when I arrived he hadn’t liked having women in the lab because they cried, but that he had changed his mind – I can’t promise that I never cried. Since then Alan has had several outstanding women in his group, and the LMB now has several women as group leaders. It was my impression that the atmosphere at Oxford was quite different, probably because Dorothy Hodgkin was there. I have often wondered how it might have been had Rosalind Franklin joined the LMB. Her premature death prevented this.

After three years I was ready to return to the United States and set out on my own. Finding a job across the Atlantic was difficult for many. It was before the requirement to advertise positions and the Internet. One heard about jobs informally, often by chance. I joined the faculty of a young and growing department at Carnegie Mellon University in Pittsburgh, Pennsylvania in 1976. I was there for nine years before moving to Robert Wood Johnson Medical School at Rutgers University in New Jersey. Working in the atmosphere at the LMB helped give me the confidence throughout my career to try new things, to be bold, to do something when I am not an expert, to do what others are not doing, to be a bit impulsive, to keep the science fun, to do things right, to not be in a rush, to try to make a difference. I haven’t always succeeded, and I have not won awards though I have managed to remain funded, and for the most part I have immensely enjoyed my work. The best part, though, is the lasting friendships that began at the LMB.

CHAPTER 32

At the LMB (1974–5)

Robert Edgar

University of California Santa Cruz, CA

I first visited the LMB in 1957. I was on my way to a postdoctoral fellowship at the Pasteur Institute with Francois Jacob. I had met Sydney Brenner a few years earlier; we were lab partners at the Cold Spring Harbor Laboratory in a course in bacterial genetics. (I remember being surprised to learn that Sydney had come from Hinshelwood’s lab at Oxford, as Hinshelwood was known for arguing that bacteria did not have genes.)

My 1957 visit to the LMB was for just a few days; two American friends, Seymour Benzer and George Streisinger, were working with Sydney on studies of bacteriophage T4, that had also been the subject of my research as a graduate student. I remember having dinner at a Chinese restaurant with them and Francis Crick. It was my first experience with the lack of central heating in Britain at that time. There was an electric heater under the table to keep our feet warm as we ate the meal. I was also astonished to see the lab. As I recall it looked like a leftover barracks from the war. They called it the Hut.

By a decade later, many phage and bacterial geneticists had switched to working on eukaryotic organisms. George Streisinger was developing the genetics study of the zebra fish, Seymour Benzer was working on the genetics of the nervous system of Drosophila, and Sydney, of course, was in the midst of developing the worm, C. elegans, as a model system for the genetic study of animals.

After my year at the Pasteur, I went to Max Delbruck’s lab at Caltech and resumed working with phage T4. During the 1960s Dick Epstein and I used amber and temperature sensitive lethal mutations to identify and characterise the essential genes of the virus, and later, Bill Wood and I used these mutants to study the assembly of the viral components.

In 1970 I left Caltech to start a new college at the University of California at Santa Cruz, Kresge College. In 1974 I stepped down as Provost of Kresge College and took a sabbatical year in Sydney’s lab at the LMB to learn how to do genetic analysis of the worm. When I arrived I was shocked to learn that there was no lab space for me. (Sydney at one point told me that people worked best in a crowded lab.) Sydney let me use J.D. Smith’s office while Smith was on leave. As for doing research, I was in a quandary about what to do. I soon learned that working on the genetics of the worm was not much different from working on the genetics of T4. All I needed were petri dishes seeded with E. coli, and a dissecting microscope, so the office was adequate. Fortunately the office was next to the lab and the lab bench nearest me was occupied by Bob Herman from Minnesota, and we chatted a lot.

Bob and I contributed a minor improvement to worm manipulation. Worms were picked up and deposited on petri dishes with a sterile stick. The sticks would swell and split so a lot of time was spent shaving them to a point. Bob turned to using a nicrome wire to pick up the worms. My contribution was to shape the wire into a curved spatula so several worms could be manipulated at once. Not a great technical achievement but one that pleased me.

Sydney and Francis shared an office. Surprisingly, their desks were in the middle of the room facing each other. Whenever I walked past and they were there, they were sitting at their desks talking to each other. They were both renowned for talking.

It was a great year for the worm and I was caught up in the excitement. John White was working out the wiring of the nervous system. John Sulston and Bob Horvitz were completing the cell lineages. (It was reputed that John took his worms home every night so as not to miss crucial cell divisions.) There was the feeling that we could really learn everything about the worm.

I decided to isolate temperature sensitive mutations in genes that control early development. In this I was successful and I happily studied them until near the end of the year, when to my dismay, I received a manuscript on this very topic from my T4 coworker Bill Wood. Bill, then home at the University of Colorado, had spent a previous year at the LMB working with Sydney. Because of Bill’s work, I never published my own.

I was in awe of this great historic university. Sydney took me to High Table at King’s. I found the ritual quite impressive, especially the port after the meal, but a bit intimidating. Hearing Monteverdi in King’s College Chapel is still a vivid memory.

There had been by then many Americans who had passed through the lab to learn about the worm, most of them having previously studied phage and bacteria. They were now scattered all over the US in different universities. When I was ready to go home at the end of the year, Sydney took me aside and told me that he wanted me to foster a community of worm workers in the United States. Bill Wood and I organised the first worm meeting at Woods Hole, and then continued it every year after that at Cold Spring Harbor. I remember telling people, at that first meeting, that we should collaborate and not compete. I probably sounded pompous and needn’t have worried. Each of them had taken a part of worm – muscle, gonads, cuticle, life cycle, development – and we were all bound together by the genome, and shared the common goal of reconstructing the organism, C. elegans. Also I started a newsletter to assist the process, The Worm Breeder’s Gazette, in which each lab could share what they were doing.

When I left the LMB I took with me a collection of worm strains which I dutifully put on my customs declaration. We arrived late at night at Seattle airport. The customs officer called the agriculture inspector. I showed them the letter Sydney had given me declaring that the nematode strains were harmless soil nematodes. The inspector was not impressed. He had to call his supervisor. The supervisor showed up the next morning and let me finally go home to Santa Cruz.

CHAPTER 33

How Do Enzymes Really Work? The Dawn of Simulating Biological Functions (1974–5)

Arieh Warshel

Department of Chemistry, University of Southern California, Los Angeles, CA

In 1963, during my second year at the Technion (Israel Institute of Technology), I became interested in understanding how enzymes can accelerate chemical reactions – sometimes by up to twenty orders of magnitude. I started an experimental project that eventually resulted in probably the first NMR measurement of the fast step in the catalytic reaction of chymotrypsin, but I did not see any reasonable clues pointing towards the origin of the catalytic effect. In fact I did suspect electrostatic effects, but my experiments showed that changing the ionic strength does not influence catalysis in a major way. At that time I took a very intimidating class in quantum mechanics learning about the so-called ‘impact parameter’, at the physics department. The class was much above the level of what we learned in chemistry, and the only part that I fully understood was the implication that asymptotic quantum mechanical wave functions (functions that describe the nature of the system at its initial and final states) can always solve any complex problem in physics. Thus, I told my classmates one day that in the distant future I was going to develop an asymptotic wave function for enzymes to understand how they work. Of course, I did not have any clue what enzymes look like and how a wave function would explain the action of enzymes – eventually it was the EVB approach (see later) that captured the asymptotic features of enzymatic reactions.

After the Technion I made an appointment with Shneior Lifson, who was at that time the scientific director of the Weizmann Institute. What attracted me was a news article that said that Lifsom was from a kibbutz (Nir David) very near to my kibbutz (Sdhe Nhaom), and the fact that he was starting to move from statistical mechanics of helix coil transitions to modelling molecules with digital computers, which was still extremely far from my interest in enzyme wave function. Although Shneior was determined not to accept any students, I convinced him with the help of my grades (that he pretended to ignore) to take me, and join his non-existent group. I moved to the Weizmann Institute in autumn 1966 and started by trying to develop what became known as the consistent force field (CFF), where the general direction was to represent molecules as balls and springs (which became known as molecular mechanics or force field approach) to reproduce energies, structures and perhaps vibrations of molecules by consistent fitting of parameters to experiments. The problem was that we had no clue how to do this. As a start I attempted to treat cyclic amides, on the way to parameterising amino acids’ potential functions, by extending the internal coordinate approach of a previous student of Lifson’s (Mordechai Bixon). However, this approach, which involves derivatives of complex interdependent transformation matrices, became basically impossible. In desperation I tried to abandon the common description of molecules in terms of bond lengths and bond angles and to move to a Cartesian coordinate description, where suddenly all the problems with analytical derivatives seem to disappear. For example, obtaining the analytical first and second derivatives needed for minimising the energy of a cyclic molecule, which was close to impossible (because each internal coordinate depends on all the other coordinates), became trivial in Cartesian coordinates. Similarly, obtaining vibrational modes, which demanded spending half a year on reading Bright Wilson’s Molecular Vibrations book and then almost hopeless programming (at least for cyclic molecules), required only the use of one simple equation in terms of the Cartesian second derivatives. At this stage I was told by Lifson that this is impossible and that I must be wrong (a situation that continued throughout my career), but fortunately the Weizmann Institute had a specialised computer called the Golem (named after the ‘robot’ from Jewish legend that helps the famous rabbi from Prague) that had a remarkable double precision. Thus I was able to obtain very accurate first and second numerical derivatives and to prove that I was on the right track in obtaining exact minima and molecular vibrations in a general molecule.

At that point I started to write a program with Cartesian analytical derivatives and force field refinement (using the numerical derivative in finding errors), stopped for a while during the Six-Day War and then moved back to the program. My progress with Cartesian coordinates convinced Lifson to let me finish my Masters degree in less than a year and to basically jump to the PhD project, considering the potential for fast progress in the general CFF direction. At that time Michael Levitt appeared, and following Shneior’s suggestion we developed together the general CFF Cartesian force field programs1 that allowed one to use molecular mechanics (MM) and to find exact local minima and vibrations of any general molecule. The program also allowed for a fully consistent refinement of the MM parameters by fitting calculated and observed properties of molecules and molecular crystals. At any rate, our CFF MM program eventually became the basis of all modern molecular simulation programs.1b Incidentally, during 1968 I also started experimenting with combining my newly developed force field method and a valence bond quantum model,2 which eventually turned out to be very useful.

Figure 33.1. Shneior, Arieh and Mike.

While still keeping enzymes at the back of my mind, I started a postdoctoral position at Harvard with Martin Karplus at the beginning of 1970, hoping to make the CFF more quantum mechanical. Indeed I succeeded in augmenting the CFF program, by retaining the spring-like description of bonds with localised electrons, but then describing by a quantum mechanical treatment the energy and forces of bonds with delocalised electrons.3 However, this still did not help me to move toward studying enzymes and, upon returning to the Weizmann Institute in 1972, I started to develop a very effective hybrid orbitals quantum program (QCFF/ALL), that represented all the atoms in a relatively small part of a molecule quantum mechanically, while representing the rest classically. I felt that this should allow me to finally make progress on my old dream of studying enzymes. At that time Mike returned from the MRC to the Weizmann Institute and I started to explore the possibility of combining my quantum mechanical model with his MM calculations on lysozyme. While still struggling with the development of my QCFF/ALL I found myself one day in the computer centre discussing the protein folding problem with Mike. The discussion turned to a strange idea of studying mechanical models of molecules on a gravitation-less spacecraft, and we suddenly came out with the idea of simplified protein models where spheres would represent amino acid side chains, and started to work on this project. This folding project also started to move in a remarkable way, and it appeared that the drastic simplifications we had suggested allowed us to fold the small protein BPTI without using an enormous number of minimisation steps. This simplified model (which was the origin of what is referred to now as ‘coarse-grained modelling’) appeared to resolve the so-called Levinthal paradox, according to which proteins have so many degrees of freedom and such an astronomical number of possible conformations that a protein could never find in a reasonable time a path for folding, without perhaps specialised nucleation processes. In fact, our simulations showed that the number of relevant coordinates is relatively small and that the protein folding process can be simulated effectively. This progress on the folding problem helped me to obtain an EMBO fellowship so that I could collaborate with Mike when he moved back to the MRC.

My time at the MRC turned out to be the dawn of several key advances in computational biophysics, with several major achievements on the frontiers of understanding biological functions and finally the culmination of some of my dreams. These advances, which I will outline below, benefited tremendously from the atmosphere at the MRC and from the discussions with different members of the lab during tea breaks.

ENZYMES, ELECTROSTATICS AND QM/MM

Upon my arrival to the MRC in autumn 1974 I started to focus on my efforts on modelling enzymatic reactions, still attempting to somehow combine my quantum model with Mike’s energy minimisation of lysozyme. More specifically, the three-dimensional structures of the enzyme lysozyme, which were resolved by D.C. Phillips and coworkers in 1966, provided the first glimpse of the structure of the enzyme-substrate complex. These breakthroughs offered enormous hope that enzyme catalysis would now be understood. For example, Phillips suggested that enzymes work by applying steric strain that pushes the substrate to a structure that is closer to the structure of the so-called ‘transition state’, where the crucial bond between the carbon and the oxygen atom in the sugar substrate of lysozyme is broken. The strain was argued to reduce the barrier for bond breaking – the activation barrier. However, as Mike pointed out, the strain idea was problematic since enzymes are flexible and it seemed clear to me that any further progress would require actually modelling the chemical reaction in the enzyme.

My first attempt to combine the computer programs resulted in ridiculously high activation energies (so the reaction would never happen) and I realised that something must be completely wrong with my modelling direction. Eventually it became clear that the work of breaking the bond between the carbon and oxygen atoms in the sugar substrate was being described incorrectly. The problem was that the bond is broken to a positive carbon and a negative oxygen (C+ O–) and that these corresponding charges must be stabilised by the electrostatic environment of the protein+ solvent system. Unfortunately, no one knew how to model such an environment, or how to model electrostatic energies in proteins or solution. After spending several months in the library of the department of chemistry in Cambridge and talking to eminent experts on electromagnetic theory, I realised that none of the textbooks can tell us much about how to model electrostatic effects correctly in proteins or solutions. Thus I turned to the approach I had learned from my experience with developing force field approaches; forget about what is in books that were written before the emergence of computers, and just go to the basic molecular level, using simplified models if needed.

Figure 33.2. My wife Tami and daughters (Merav and Yael) in Cambridge, 1975.

My observation was that we would be completely unable to progress consistently as long as we thought in terms of the standard electrostatic theory, where all the details of the protein or the surrounding solvent are included with an ambiguously defined dielectric constant. Obviously, with the computer power of that time we could not use a model with a protein surrounded by fully atomistic models of water molecules and obtain meaningful energetics. Thus we represented the water molecules as a grid of polarisable dipoles (that follow the Langevin gas phase polarisation law) and solved self-consistently the interaction of the dipoles with the charges in the protein-substrate system and with each other. Similar self-consistent treatment was then introduced for the protein-induced dipoles.4 The simplified water model led to the first microscopic description of protein electrostatics, thus escaping all the conceptual traps of future continuum descriptions. However, this model looked strange to those who were trained with the idea that the special, highly symmetric structure of water molecules must be highly relevant to their enormous solvation effects. This led to interesting discussions with Aaron Klug, who felt that water molecules must have a specialised structure, whereas I tried to point out that packing small coins around larger coins cannot produce a perfect structure. At any rate, the water grid model turned out eventually to be an excellent approximation for studying solvation effects, long before any other microscopic model and also before the development of macroscopic models that tried to consider the protein shape.

The introduction of a realistic electrostatic model for the enzyme and its surrounding water molecules, together with the incorporation of this effect in a quantum Hamiltonian in what is called nowadays hybrid quantum mechanical/molecular mechanics (QM/MM),4 led for the first time to a reasonable energy for the bond breaking process in enzymes and solutions. This QM/MM approach exploited Einstein’s advice to ‘make everything as simple as possible, but not simpler’. That is, in this case we realised that we cannot treat large systems quantum mechanically and we cannot describe the chemistry without quantum treatment. Thus we decided to describe only the reacting region quantum mechanically, while treating the rest of the protein and the solvent classically. This approach, along with related methods that I introduced subsequently, has become known as ‘multiscale modelling’. The QM/MM research suggested that enzymes work by using electrostatic fields to reduce bond breaking activation barriers. At any rate, the modelling of the catalytic reaction of lysozyme using our approach paved the way for the current direction in modelling enzyme action and to the enormous explosion in QM/MM simulations of enzymes5 which have become a major direction in theoretical chemistry and biophysics. The QM/MM studies also led to what I believe is a true understanding of the origin of enzyme catalysis, which turned out to be associated with the electrostatic preorganisation effect.6 More specifically, my subsequent study led to the non-trivial finding that enzyme catalysis is not due to the interaction between the enzyme and substrate (which is what was believed by most people) but to a large free energy penalty for the reorganisation of the solvent in the reaction without the enzyme (the work of rotating the water molecules towards the transition state charges). This reorganisation energy increases the activation barrier in solution, whereas the enzyme polar groups that stabilise the transition state do not have to rotate, since they are already folded with correctly polarised dipoles. In subsequent years I was also able to prove that the change in the electrostatic reorganisation energy accounts for almost the entire catalytic power of enzymes. Although this elusive origin had not been envisioned during our studies at the MRC, I had benefited from the general feeling that it should be associated somehow with electrostatic effects (see Max Perutz’s insightful review7). In fact my identification of the electrostatic preorganisation effect led to long communication with Max who agreed to communicate this work to PNAS. However, while Max recognised intuitively the importance of this effect he felt originally that it was like the assumed stabilisation of ion pairs in a low dielectric environment, while I found that enzyme active sites are in fact very polar, and pointed out to him that ion pairs would not be stable in oil surrounded by water; this explanation led eventually to another paper that he communicated for us.8

Figure 33.3. The QM/MM model of an enzyme active site. The enzyme (trypsin) is divided into a small reactive QM region and the rest of the system which is described classically.

The electrostatic models conceived in the MRC days have become the basis for consistent microscopic treatments of biological molecules, the understanding of the true nature of protein dielectric constants9 and the simulation of key functional properties including: evaluation pKa values, calculations of redox potentials, evaluation of binding free energies, simulation of ion and proton conductance and even evaluation of protein stabilities.10

In subsequent years, I simplified the QM/MM approach using a valence bond description of the different steps of the reaction in what I called the ‘Empirical Valence Bond’ (EVB) method. This method, which can be related to my strange 1963 asymptotic wave function idea, has finally allowed me and my coworkers to take a considerable leap towards approaching my early vision, and to finally model quantitatively enzyme catalysis and explore enzyme design. This also helped me to explore and frequently to eliminate popular suggestions of factors that presumably lead to enzyme catalysis (e.g. entropic effects, ground state destabilisation by desolation, dynamical effects, orbital steering and more). The key to the ability to figure out the secret of enzyme catalysis has been the ability to model the actual chemical reaction in the enzyme active site and to dissect the contributions to the rate constant, which is close to impossible when one is just using experimental approaches.

THE PRIMARY EVENT IN VISION AND THE DAWN OF MOLECULAR DYNAMICS SIMULATIONS IN BIOLOGY

Before arriving at the MRC I realised that the best and perhaps the only way to study photochemical reactions was to forget about the traditional description of crossing between energy levels and to adopt the surface hopping semi-classical trajectory approach that was introduced for treating gas phase reactions of very small molecules.11 My conceptual breakthrough occurred in 1971 and was published eventually in 1975,12 but without any direct relationship to biology. The fact that the MRC dealt with real biological systems inspired me to try to use this approach in looking at the primary event in the visual process. That is, at that time it was known that when light strikes the eye, it is absorbed in a Schiff base of retinal, which is embedded in a protein called rhodopsin. After absorption of light the retinal molecule isomerises from its initial 11-cis structure to an all-trans structure, forcing structural changes of the protein, where the new form of the protein (metarhodopsin) activates the transfer of the visual signal to the brain. Later, it was found that the metarhodopsin activates a G protein called transducin. It was also known that the primary absorption of light leads to a photoisomerisation of the retinal molecule in less than six picoseconds (which was the shortest time that could then be measured). However, it was not clear what was actually happening and how one could simulate this key biological step. Furthermore, the absence of structural information seemed to introduce even a bigger challenge. Thus, I had the ‘brilliant’ idea of binding retinal to chymotrypsin, obtaining crystals and measuring the quantum yield. However, when I suggested this project to Richard Henderson, who was working at the MRC on the structure of bacteriorhodopsin, he declined, saying that in his early work with chymotrypsin he developed an allergy to this protein and could no longer touch it. Thus I decided to model the protein effect by a steric cavity plus an assumed internal counter ion, and used the semiclassical surface hopping approach with a Schiff base of retinal constrained to be in the starting 11-cis conformation.13 My molecular dynamics (MD) simulations predicted that the primary process takes about 100 femtoseconds, with an enormous probability of jumping to the ground state due to very large non-adiabatic coupling (a phenomenon that was later identified as the effect of conical intersection). Remarkably the results of these simulations, that represented the first use of MD simulations in biology, have since been confirmed both experimentally14a and theoretically.14b

Figure 33.4. Snapshots from the simulated MD trajectory of the primary event in vision. The trajectory starts with 11-cis retinal in the ground state and upon absorption of light moves to the excited state, where the torsional angle of the 11-12 bond (shown in a cis configuration in the left figure) rotates without a barrier to 90 degrees and the trajectory crosses to the ground state in the trans direction. The motion involves only a small change in overall structure, since other torsional angles move in the opposite direction to the 11-12 angle. The simulated time for moving from the initial excited state to the crossing point is around 50 femtoseconds. The red point represents a negatively charged group of the protein that interacts with the positively charged nitrogen of the Schiff base. The snapshots are taken from a movie that used the original trajectory presented in reference 13.

The molecular motion that emerged from these computer simulations resolved the problem of moving fast in a restricted protein cavity without strongly clashing with it. That is, it was found that the isomerisation occurs with a concerted rotation of several bonds, which I called the ‘bicycle pedal’ motion. To see if the bicycle pedal model made sense I borrowed model building parts from Max’s structural biology lab and built a model that appeared to reproduce the concerted motion without any large structural change. Interestingly, this motion has been confirmed after about 30 years by ab initio studies.14b The following years15 I used the structure of a bacterial reaction centre and the same semiclassical approach to model the primary electron transfer event in photosynthesis, establishing that the observed 3 ps process involves a sequential hopping from the primary chlorophyll dimer (P*) to one monomer (B) and then to a second monomer (H). This was done again before the confirmation of our findings by decisive experimental studies, and at a time where most workers assumed that the primary event is not a sequential process but a superexchange process. Here, the ability to determine the correct electrostatic energy of each intermediate has been a major advantage over related attempts that did not involve experience in the conversion of protein structures to electrostatic energies and redox potentials.

Haemoglobin

Another important direction, that emerged from talks with Max Perutz, was the effect of the heme on the nature of the allosteric effect in haemoglobin. At this time it was not entirely clear what is going on when oxygen binds to the iron ion at the centre of the heme group of haemoglobin. The prevailing story (that Max liked) was that when oxygen binds to the iron, the heme must change from a non-planar high spin structure to a planar low spin structure. This spin transition, which on a side-note explains the different observed colours of arterial and venous blood, was thought to be the trigger for the allosteric transition of haemoglobin. This meant that sophisticated quantum mechanical effects tell haemoglobin what to do. However, the quantum mechanical force field (that included the effect of iron orbitals) that I developed at the MRC with the help of my then PhD student Andrea Lapiccirella, helps me to explore the actual reason why the heme in haemoglobin becomes planar.16 We found that this has very little to do with the change in spin state but is just due to the fact that when the iron has two axial ligands (the protein histidine and the bound oxygen) they are both repelled by the heme nitrogen atoms and the competition leads to a planar structure. This daring idea was successfully confirmed by an experiment when it was found that two axial ligands force even a high spin heme to become planar. This is why I was later quoted as saying that ‘haemoglobin never learned group theory’.

Using CG approaches in modelling the action of molecular machines

Although I continued during my MRC days to extend the simplified folding model to studies of helical proteins, I have not put significant effort into this model for almost the following two decades (while the use of this and related types of simplified models have exploded in studies of the protein folding problem). This is because my interest has been in protein function. However, I eventually augmented this CG model with specialised electrostatic terms, and found it to provide what is arguably the best current tool for moving from structure to function of molecular machines.10 This model allowed us to simulate17 the vectorial action of F1-ATPase (which is arguably the best-studied biological motor), the conversion of proton gradient to work in F0-ATPase,18 the action of voltage activated ion channels19 and the function of other molecular machines.

SO WHAT WAS THE ROLE OF THE MRC IN MY SCIENTIFIC VOYAGE?

My interest in enzymes and biology started before I joined the MRC , and some of the key advances began before then. However, coming to the lab in 1974 gave me a very different perspective; suddenly I found myself in an environment where the absolute frontier of structural biology was the subject of every day, every tea break, and every discussion. This led to remarkable progress in just under two years, when Mike and I produced several groundbreaking works that paved the way for a large part of what is now called ‘Structural and Functional Computational Biology’. This includes the first solution of the folding problem, the introduction of what is now called computational enzymology, the emergence of molecular dynamics simulations of biological functions and the foundation of microscopic electrostatic studies of proteins, as well as the emergence of calculations of spectroscopic properties of proteins.20 This fruitful period has guided me in my future research and in deciding that if you ask the right question, it always seems possible to eventually construct an effective model for the detailed workings of highly challenging, real biological systems. The realisation that computer simulations provide us with powerful means to understand biological functions has helped me and kept me going throughout my career, but nothing could compete with the fast progress and the inspiration of my remarkable time at the MRC.

REFERENCES

1. (a) S. Lifson and A. Warshel, 1968. A Consistent Force Field for Calculation on Conformations, Vibrational Spectra and Enthalpies of Cycloalkanes and n-Alkane Molecules, Journal of Chemical Physics 49, 5116. (b) M. Levitt, 2001. The birth of computational structural biology 8, 392.

2. A. Warshel and A. Bromberg, 1970. Oxidation of 4a, 4b-Dihydrophenanthrenes. III. A Theoretical Study of the Large Kinetic Isotope Effects of Deuterium in the Initiation Step of the Thermal Reaction With Oxygen, Journal of Chemical Physics 52, 1262.

3. A. Warshel and M. Karplus, 1972. Calculation of Ground and Excited State Potential Surfaces of Conjugated Molecules. I. Formulation and Parameterization. Journal of the American Chemical Society 94, 5612.

4. A. Warshel and M. Levitt, 1976. Theoretical Studies of Enzymatic Reactions: Dielectric Electrostatic and Steric Stabilization of the Carbonium Ion in the Reaction of Lysozyme, Journal of Molecular Biology 103, 227.

5. S.C.L. Kamerlin, M. Haranczyk and A. Warshel, 2009. Progresses in Ab Initio QM/MM Free Energy Simulations of Electrostatic Energies in Proteins: Accelerated QM/MM Studies of pKa, Redox Reactions and Solvation Free Energies, Journal of Physical Chemistry B, Centennial Feature Article 113, 1253.

6. A. Warshel, 1978. Energetics of Enzyme Catalysis. Proceedings of the National Academy of Sciences of the United States of America 75, 5250.

7. M.F. Perutz, 1978. Electrostatic effects in proteins. Science 201, 1187.

8. A. Warshel, S.T. Russell and A.K. Churg, 1984. Macroscopic Models for Studies of Electrostatic Interactions in Proteins: Limitations and Applicability, Proceedings of the National Academy of Sciences of the United States of America 81, 4785.

9. A. Warshel and S.T. Russell, 1984. Calculations of Electrostatic Interactions in Biological Systems and in Solutions. Quarterly Review of Biophysics 17, 283.

10. S.C.L. Kamerlin and A. Warshel, 2011. Multiscale Modeling of Biological Functions. Physical Chemistry Chemical Physics 13, 10401.

11. J.C. Tully and R.M. Preston, 1971. Journal of Chemical Physics 55, 562.

12. A. Warshel and M. Karplus, 1975. Semiclassical Trajectory Approach to Photoisomerization. Chemical Physics Letters 32, 11.

13. A. Warshel, 1976. Bicycle-pedal Model for the First Step in the Vision Process. Nature 260, 679.

14. (a) Q. Wang, R.W. Schoeniein, L.A. Peteanu, R.A. Mathies and C.V. Shank, 1994. Vibrationally Coherent Photochemistry in the Femtosecond Primary Event of Vision. Science 266, 422; (b) L.M. Frutos, T. Andruiow, F. Santoro, N. Ferre and M. Ollivocci, 2007. Tracking the excited-state time evolution of the visual pigment with multiconfigurational quantum chemistry. Proceedings of the National Academy of Sciences of the United States of America 104, 7764.

15. S. Creighton, J.-K. Hwang, A. Warshel, W.W. Parson and J. Norris, 1988. Simulating The Dynamics of the Primary Charge Separation Process in Bacterial Photosynthesis. Biochemistry 27, 774.

16. A. Warshel, 1977. Energy-Structure Correlation in Metalloporphyrins and the Control of Oxygen Binding by Hemoglobin. Proceedings of the National Academy of Sciences of the United States of America 74, 1789.

17. S. Mukherjee and A. Warshel, 2011. Electrostatic Origin of The Mechanochemical Rotary Mechanism And The Catalytic Dwell of F1-ATPase. Proceedings of the National Academy of Sciences of the United States of America 108, 20550.

18. S. Mukherjee and A. Warshel, 2012. Realistic simulations of the coupling between the protomotive force and the mechanical rotation of the F0-ATPase. Proceedings of the National Academy of Sciences of the United States of America 109, 14876.

19. A. Dryga, S. Chakrabarty, S. Vicatos and A. Warshel, 2012. Realistic Simulation of The Activation of Voltage-Gated Ion Channels. Proceedings of the National Academy of Sciences of the United States of America 109, 3335–40.

20. A. Warshel, 1977. Interpretation of Resonance Raman Spectra of Biological Molecules. Annual Review of Biophysics and Bioengineering 6, 273.

CHAPTER 34

A Cell Biologist’s Explorations in a Molecular Biologist’s Paradise (1976–7)

J. Richard McIntosh

Department of MCD Biology, University of Colorado, Boulder, CO

Figure 34.1. J. Richard McIntosh.

In the mid-1960s I was a graduate student at Harvard, working in the lab of Keith Porter. Keith was an early and important student of microtubules, which his electron microscopy (EM) had found in numerous cells and tissues. He surmised that these cytoplasmic fibres were girders that helped to define and maintain cell structure, so all his students were told to find a tissue in which cells were asymmetric and see by EM whether microtubules (MTs) were distributed in ways that might contribute to cellular morphogenesis. I undertook the process of spermiogenesis, i.e. how an essentially spherical spermatocyte metamorphoses into the highly asymmetric form of a mature sperm cell, a process essential to the reproduction of most animals and plants. I started with insects, based on light microscope images of sperm drawn beautifully by Gustaf Retzias. Studying these cells by EM was rewarding, because they were full of MTs, but I aspired to learn something about how MTs worked, so I wanted a source of material that was suitable for biochemistry as well as imaging. I settled on rooster testes as an ample and convenient source of cells that were developing asymmetries. Following Keith’s guidance, I used EM to describe the shape changes that occurred as the spherical rooster spermatocytes transformed into cylindrical spermatids. My images showed that the elongating nucleus was surrounded by a double helix of MTs that seemed to act as a cinch, constricting and elongating the sphere (McIntosh and Porter, 1967). These observations define two problems of great interest (to me): how was the assembly of this helical MT array controlled and how did the turns of the helix slide over one another to effect nuclear constriction? There were at least two obvious approaches to these questions. One was the biochemistry of MTs, where the only assay with which to follow their purification was their shape as seen in the EM, a Herculean task on which I made little progress. The second approach was better imaging of the MTs and the tiny bridges that connected the adjacent helical gyres.

At this time the structural studies group at the Laboratory of Molecular Biology (LMB) at Hills Road, Cambridge, included a few senior scientists with great skill in EM (e.g. Hugh Huxley) or in the analysis of EM images (e.g. Aaron Klug). There was also a wealth of younger people doing spectacular work on the structure of viruses (Tony Crowther, John Finch, David DeRosier) and a variety of cellular structures (Linda Amos, Richard Henderson and Nigel Unwin). Members of this group mounted short but intense courses in ‘high resolution EM’, and I had the pleasure of attending one of the first (the summer of 1968, I believe). This exposed me to an entirely different way of thinking about the EM and its ability to reveal details of biological structure. It was my first exposure to methods for high quality negative staining, to diffraction as a way to make reliable observations on the average structure in a periodic array, to optical filtering, and more generally to an analytic approach to structure. It got me thinking about the structural polarity in MTs and how this might affect the behaviour of the MT-associated motors that seemed to inhabit cytoplasm. These ideas were, of course, heavily influenced by the sliding filament ‘theory’ of muscle contraction, which had by this time been fully established. They were also influenced by the structural studies of Peter Satir, which had demonstrated MT sliding in cilia (Satir, 1963), and the discovery by Ian Gibbons of dynein, an ATPase in cilia and flagella (Gibbons and Rowe, 1965). This context motivated me and my young colleagues at Harvard to develop a model for mitosis, following the then-common paradigm that one should seek simple models for complex biological processes. We found that there was one, and only one, remarkably simple set of postulates about MT polarity and motor enzyme action that would explain a huge amount of otherwise disconnected phenomenology on chromosome segregation, using only a single MT-sliding motor. With a helpful nudge from Aaron Klug we were able to publish this model in Nature (McIntosh et al, 1969), so we set about testing it.

One aspect of the model was the idea that MTs in the cytoplasm could slide over one another as a result of bridges analogous to dynein or the myosin cross-bridges between thick and thin filaments in muscle. Our first effort to understand such motors was to study a highly ordered array of MTs called the ‘axostyle’ of a flagellated, symbiotic protozoan, Saccinobacculus. In collaboration with others in Porter’s lab, we got high quality electron micrographs of thin sections cut from two species of this protozoan and used optical diffraction to follow the relative positions of its component MTs and their cross-bridges during the formation and propagation of bends along the axostyle (McIntosh, 1973; McIntosh et al, 1973). This work, which built on some of the training in image analysis that I got during my first visit to the LMB, suggested that the MTs of this almost crystalline cytoplasmic fibre did indeed slide relative to one another as the axostyle wiggled, a proposition supported by the discovery that isolated axostyles contained dynein (Mooseker and Tilney, 1973).

Another activity that followed on from the LMB structure course was our efforts to develop assays for the structural polarity of cytoplasmic MTs; these would have allowed us to test both the assumptions and some predictions of our model for mitosis. At the time, there was no way to visualise MT polarity, because EM images of cytoplasmic MTs were not of sufficient resolution to display the polar orientation of its subunits, the dimeric protein tubulin. Linda Amos had, however, been successful in seeing subtle differences in the orientation/position of alpha and beta tubulin in the walls of flagellar MTs, using high quality negative staining and optical image processing (Amos and Klug, 1974). I arranged a summer visit to the LMB to try to learn these methods well enough to see structural polarity in the simpler MTs that form in cytoplasm. In 1968, Ed Taylor’s group at the University of Chicago had published a series of landmark papers on the identification of the protein subunit of tubulin (what I had hoped to do by MT isolation), and shortly thereafter, Richard Weisenberg, one of those talented students who was now working independently, discovered a way to polymerise tubulin in vitro (Weisenberg, 1972). It was therefore practical to make cytoplasmic MTs in quantity and see if we could visualise their polarity by appropriate analysis of EM images of negatively stained samples.

Linda and Nigel Unwin kindly helped me with this project for a summer, but we couldn’t get images that revealed MT polarity with any certainty. My chief recollection of that time was Linda’s kind but measured response to my enthusiasm. I would take my best images of MTs, digitise them and compute their Fourier transforms, then point enthusiastically to the spots that were just visible on the 2 nm layer-line, hoping that these would include the information we needed to see polarity-revealing detail. Linda would smile, but point out that those spots were ‘a bit dubious’. It was only some years later that a chance discovery by Steve Heidemann, then working as a postdoc in my lab, gave us a method for the overt display of MT polarity by decorating existing MTs with exogenous tubulin, assembled onto the cellular polymers under non-physiological conditions (Heidemann and McIntosh, 1980). That innovation allowed Ursula Euteneuer in our lab to visualise MT polarity in the MTs of several mitotic spindles (Euteneuer et al, 1982; Euteneuer and McIntosh, 1980; Euteneuer and McIntosh, 1981; McIntosh and Euteneuer, 1984). These data demonstrated authoritatively that the postulates and predictions of MT polarity in our spindle model were wrong. So much for simple ideas about complex biological processes!

During the academic year 1976–7 I returned to the LMB for a full sabbatical year. My lab was still quite firmly focused on understanding chromosome motion, but I wanted to explore other aspects of cellular machinery, for example the ways in which MTs might contribute to morphogenetic events during animal development. Sydney Brenner was kind enough to accept me as a visitor at a time when his lab was making huge strides in understanding the development of C. elegans. I flirted with doing serious developmental genetics, along with Bob Horvitz, Bob Waterston and Marty Chalfie who were then postdocs in the lab. I also thought about joining John White and Nichol Thomson in their EM study of the nematode nervous system, but I turned instead to a more conservative course of action: looking in some detail at mitosis in the post-embryonic development of the nervous system. I learned a large amount, thinking with these talented people about the use of genetics to tackle complex cellular problems in the background of a whole organism, but as I grappled with the practicalities of finding informative mitotic mutants in the context of a multicellular organism, I became convinced that I should work with a simpler beast. This year also included many teatime conversations with Mark Bretscher about the role of lipid flow in cell motility. He was adamant that Gerry Edelman’s emphasis on the role of cytoskeletal machinery in membrane patching and capping was nothing but ‘Edelmania’. I confess that the net effect of these conversations was to nurture my long-standing faith in the importance of cytoskeletal fibres and motor enzymes for the mechanical processes that cells could accomplish.

An obvious choice for a genetic approach to mitosis was budding yeast, but even in this system, the complexity of mitosis made it far from obvious how to use genetics to identify interesting mitotic molecules, like MT-associated motor enzymes. Since broad-ranging genetic analyses of yeast cell division were already well advanced in the labs of several great geneticists, I decided to try to find a yeast mitotic motor by biochemistry. John Kilmartin was kind enough to accept me as a visitor for my next sabbatical leave, so in 1983–4 I again visited the LMB with the goal of using affinity columns, based on tubulin isolated from mammalian brain, to seek MT-interacting proteins that might be significant for mitotic mechanisms in yeast. With the invaluable help of Jan Fogg, I grew and ground up large numbers of yeast cells, made cytoplasmic extracts, and ran them through tubulin affinity columns. The idea was that the proteins which bound to column-associated tubulin but could be eluted with MgATP would include motor enzymes. This effort produced bands on gels, which John kindly used to make monoclonal antibodies, but we never got any spindle-specific staining. I did, however, try the same methodology on extracts from nematode embryos, taking advantage of my earlier friendships with Sydney’s group. This approach was strikingly successful. We found several MT-binding proteins that were released by ATP, and one of these had the unusually high molecular weight of dynein’s ‘heavy chain’. Moreover, it showed the unusual property, characteristic of this polypeptide, of being cleaved at one specific site when irradiated with UV light in the presence of ATP and vanadate ions. This work allowed our lab to be one of the first to find cytoplasmic dynein and to study its roles in cellular mechanics (Lye et al, 1987). Subsequent work, based on these advances, let us learn that dynein localised at the kinetochores of mitotic chromosomes (Pfarr et al, 1990). Then Michael Koonce, a postdoc in the lab, went on to identify sequences from the dynein gene of Dictyostelium discoidium. He and Paula Grissom ultimately cloned and sequenced this entire 14 kb open reading frame, providing the tools necessary to explore dynein function in cells that never made cilia or flagella (Koonce et al, 1992). Polypeptides expressed from these DNA sequences provided function-blocking antibodies to dynein that let us explore its possible roles in chromosome motion, and to identify its role in spindle pole separation (Vaisberg et al, 1993).

It is clear from the above that the LMB has had a great influence on the science I have been able to do; both the methods and the information I learned there were invaluable. However, the style and mindset of the LMB were equally important. There was something in the atmosphere at Hills Road that exuded quality of thought, care in execution, and energy in pursuit of excellence. It also reflected the power of intelligent collaboration between labs with different kinds of expertise. Several of my colleagues in our department at the University of Colorado were also blessed with formative visits to the LMB, so it was comparatively easy for the attitudes of the LMB to seep into the ways in which our group developed.

I had come to Boulder in 1970 as a new department of modern biology was just taking shape. Talented scientists, who had been in Boulder for some years, wanted to form in a broadly focused department of modern biology. This group included some cell biology luminaries, like David Prescott and Lester Goldstein, as well as some well-established developmental biologists; they recruited Keith Porter as chair. Together, they were able to obtain a ‘Health Sciences Advancement Award’ from the NIH, which not only built us a new building on Boulder’s beautiful main campus, but also equipped many labs, including my own. The scientists already there recruited Peter Kuempel, who had been studying bacterial DNA replication and cell cycle at Princeton, Larry Gold and Charlie Yegian, who were working on the biology of T4 phage and its interactions with E. coli. Howard Berg, then studying bacterial chemotaxis, and three structural cell biologists (Andrew Staehelin, Mary Bonneville and myself) came with Keith from Harvard, drawn by the excitement that pervaded a new effort of this kind. Then David Hirsh came from the LMB, where he had just finished a postdoctoral stint with Sydney Brenner, working on nematode development. We all thought long and hard about how to make a scientific atmosphere that would match the best that we had experienced elsewhere.

The rather intangible aspects of mood and attitude about scientific research that pervaded the LMB had a big effect on the scientific climate that developed in Boulder. Part of this was simply the image we had of high quality work. Part was the spirit of collaboration that both David Hirsh and I had experienced at the LMB. One example of this was the way nucleic acid sequencing on the third floor interacted constructively with the generation of mutant tRNAs on the second floor. Another was the way the structure groups would share instruments, computers and even reagents to make projects move forward quickly. Our group in Boulder talked a lot about how we might nurture a similar atmosphere. While the tyranny of NIH grants and their accounting made it impossible to have anything like the ‘Stores’ of the LMB, we could share instruments and expertise. I learned a lot about genetics and animal development from Hirsh’s worm group, while he learned from me about light and electron microscopy and their value for studying worm development. When Bill Wood joined the department, bringing both another T4 lab and a new group studying nematode development, the flow of ideas and technologies between the department’s multiple groups was greatly enhanced. Moreover, David’s friendship with Sydney contributed to that notable scientist coming to Boulder several times to give lectures that set everyone on his/her ear, given Brenner’s inimitable style of thought and lecturing. He introduced us to his new sense that ‘T4 is for little boys’ and got us thinking about animal development as a counterpoint between the American Plan (where your fate is destined by your neighbours) vs. the European Plan (where lineage is all). It was an exciting time, and it led to both some excellent science and a significant number of well-trained, talented students who went on to do great work in their own right. These were Boulder’s intellectual progeny, who were in some sense grandchildren of the LMB. So the legacy of the LMB is deep, broad and multidimensional. It is a place that has left a positive mark on much of the biomedical science all over the world for the last 50 years. What an achievement for the lab’s participants to reflect on!

REFERENCES

Amos, L. and A. Klug, 1974. Arrangement of subunits in flagellar microtubules. Journal of Cell Science 14, 523–49.

Euteneuer, U., W.T. Jackson and J.R. McIntosh, 1982. Polarity of spindle microtubules in Haemanthus endosperm. Journal of Cell Biology 94, 644–53.

Euteneuer, U. and J.R. McIntosh, 1980. Polarity of midbody and phragmoplast microtubules. Journal of Cell Biology 87, 509–15.

Euteneuer, U. and J.R. McIntosh, 1981. Structural polarity of kinetochore microtubules in PtK1 cells. Journal of Cell Biology 89, 338–45.

Gibbons, I.R. and A.J. Rowe, 1965. Dynein: A Protein with Adenosine Triphosphatase Activity from Cilia. Science 149, 424–6.

Heidemann, S.R. and J.R. McIntosh, 1980. Visualization of the structural polarity of microtubules. Nature 286, 517–9.

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CHAPTER 35

Lizards, Claret, Ribosomes: my PhD at the MRC-LMB (1977–81)

Werner Kühlbrandt

Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

Figure 35.1. Paul Wagner, Werner Kühlbrandt and Jon Scholey.

It was all because of Max. Max Perutz had visited Berlin in the winter of 1976–77 to give a seminar at the Max Planck Institute of Molecular Genetics, at the invitation of its director, Heinz-Günter Wittmann. Wittmann was the prototype of an old-style German professor and Max Planck director, who ruled his massive department with a firm hand and an iron will. I had started work towards a diploma thesis in his laboratory. During my entire six months there he addressed me twice in his low, ponderous, East Prussian voice. Diploma students evidently did not get much of the great man’s attention. My group leader was Roger Garrett, a bearded English, liberal, probably left-wing biochemist. Roger was openly critical of his director, whom he regarded as an authoritarian monster. This was Berlin not so many years after the 1968 student revolt, which still reverberated at the nearby Free University, the epicentre of intellectual unrest where I had studied.

RIBOSOMES IN BERLIN

Wittmann was a world authority on ribosomes, and his department was, by a very long margin, the best place in Berlin for biochemistry research at the time. And biochemistry research was what I wanted to do, preferably on ribosomes. Even at the tender age of 25 I knew that whoever solved the structure of the ribosome would win the Nobel Prize. Not that it was at all likely this would involve me in any way – I did not think I would live long enough to see the structure (which of course has now been solved and the Nobel Prize duly awarded to Ada Yonath, Tom Steitz and Venki Ramakrishnan in 2009). No more did I think that I would ever become a Max Planck director myself – Wittmann, for all his admirable determination to crack the ribosome, was not my role model. Nobody, least of all myself, would have predicted that this lay ahead of me twenty years later.

My diploma project was not going well. I was supposed to prepare and characterise an RNA-protein core in the large ribosomal subunit of E. coli. So I isolated large subunits and treated them with increasingly savage amounts of protease. The result was always a tiny, sticky pellet at the bottom of an Eppendorf tube, whereas what I wanted was crystals. Not of the ribosome – that was surely impossible, a ribosomal protein would do – but there were at the time no protein crystallographers at the institute, or anywhere in Berlin. (Wittmann recruited Ada Yonath only one or two years later and she did crystallise the large subunit – but I had left by then.)

A few months into my diploma work I had written to the ‘Head of Department, MRC Laboratory of Molecular Biology, Cambridge’, to ask about the possibility of a PhD project there. In 1974 I had won a one-year scholarship to King’s College London, which offered an excellent course in biophysics and biochemistry, a most welcome break from the chemistry course at the Free University (although this was also excellent). At King’s it was impressed on me that the best work in biochemistry and biophysics was going on at a particular laboratory in Cambridge, called the MRC-LMB, which shaped my resolve to go there, if possible, for my PhD. This was why I wrote, but I was unaware, and too naïve to find out, to whom I was addressing myself.

To my surprise and delight I received a very kind reply from Max Perutz who wrote that he was about to visit Berlin to give a lecture at the Max Planck Institute, and why not meet for a chat after his talk. What a difference! I went to Max’s lecture in the MPI auditorium with a sense of intense anticipation. It was the first of several I heard him give on the structure of haemoglobin and its amazing allosteric mechanism of oxygen-binding – all of them lucid, inspiring, and radiating with contagious enthusiasm for this protein and, in general, his life’s work, protein crystallography. I was too excited to remember much about this particular talk, but when I went up to meet him afterwards, he suggested a walk, which I remember extremely well.

It was a frosty, clear January night. Max wore his winter coat and a woolly hat with a bobble on top. I did not know that he had been a keen mountaineer and skier, and that this was probably his Austrian skiing hat. As we were walking the tree-lined avenues of Berlin Dahlem around the institute, he told me about some of the work in the Division of Structural Studies at the LMB. Much of this must have been to do with protein crystallography, but what sticks most clearly in my mind is that he mentioned some remarkable work by two of his colleagues who had determined protein structures by electron microscopy. I barely knew what an electron microscope looked like. At the biophysics course at King’s, we had been lectured on physical methods by none other than Maurice Wilkins, who had shared the Nobel Prize for the DNA structure with Watson and Crick. As one of the laboratory practicals at King’s, I had recorded X-ray diffraction patterns of self-drawn DNA fibres with a camera chamber that looked a bit like a circular sandwich tin, and for a student project I had solved the X-ray structure of a nucleotide in an unusual conformation. So I knew about X-ray crystallography, small-angle X-ray scattering, even neutron diffraction. But electrons? ‘Would this have been small-angle electron scattering?’ I ventured. No, Max replied, he did not think the angles were particularly small. (They are actually exceedingly small, due to the short electron wavelength, but neither he nor I realised this, and it was not the point.)

A few weeks later I got another friendly letter from Max, saying that, yes, I would be welcome to do my PhD in the Division of Structural Studies at the LMB. There was a young man working on ribosome structure by electron crystallography who might be in need of a PhD student. Would I be interested? Indeed I was. Max went on to write that as a PhD student I would have to register with Cambridge University and choose a college. His own college, Peterhouse, he wrote, was not particularly strong in science. Some of his students had been very happy at Churchill or Trinity. The concept of a college meant absolutely nothing to me and as it turned out, could not have been further from student life in West Berlin. So after another few months the registration papers arrived, with a list of colleges, each with a box to tick next to it. I looked down the list, and the name ‘Peterhouse’ rang a vague bell – so I ticked it. I knew nothing about the college – these were the days before the Internet – nor did my choice seem to be in any way important. Duly I was admitted by Peterhouse.

CLARET AT PETERHOUSE

Little did I know what I had let myself in for. Peterhouse was one of the last all-male colleges in Cambridge and as a result had become a stronghold of unbridled male homosexuality. Incongruously, it was also a stronghold of right-wing conservatism. Maurice Cowling, the Senior Tutor, was the political advisor of Margaret Thatcher, the right-wing prime minister. Right-wing philosophers and historians were invited to give seminars. There were rumours that some Fellows wore black armbands on the anniversary of General Franco’s death (I never saw this), and that one of the fellows was the illegitimate son of Martin Bormann, the Nazi horror, as the undergraduates gossiped not without pride. All this contrasted oddly with a set of senior fellows that included some of the most distinguished scientists in Cambridge, notably Max Perutz and Aaron Klug, who were enlightened, liberal family men of Jewish origin. They were, however, rarely seen around the college, and as far as I remember never at High Table. I sometimes wondered how much Max was aware of all this. After a few months, when I was well settled into my college flat at 17 Fitzwilliam Street, Max came into my office and asked ‘Are you having a gay time at Peterhouse?’ My jaw dropped. Was my unconditional devotion to the opposite sex that difficult to tell? Or was it possible that this kind man was unaware of the change in meaning of the word ‘gay’ since he had first arrived in England in the late 1930s? I cannot remember what I said. We both looked at each other and laughed.

As it happened, I was having a wonderful – although not exactly gay – time at Peterhouse. Its graduate society, of which I was automatically a member, provided an instant community of bright budding academics from a wide range of backgrounds, who openly welcomed me as a newcomer. There were few if any other scientists, but I met historians, engineers, language scholars and architects. What a change from Berlin, where virtually all the academic contact I had was with chemists. There was a graduate table in Formal Hall, which I attended quite regularly, even though I had to wear a gown – a long black graduate gown, not the bat-like outfit worn by the undergraduates. This would have been completely unthinkable anywhere in Berlin or Germany, and added a sense of exotic attraction. Of course the main benefit of the graduate table was that I did not have to cater for myself in the evenings, and that there was always congenial company and interesting conversation. The food was very decent – Peterhouse prided itself on having the best kitchen, and one of the best cellars in Cambridge. The latter was not really intended for mere mortals such as graduate students, but one could buy a reasonable bottle at a very reasonable price at the buttery to have with dinner. The favourable exchange rate of my German PhD fellowship enabled me regularly to share a bottle with friends around the table, and perhaps this explains why I was never short of company or good conversation in Formal Hall. Several of the other regulars became lifelong friends, including Rab Houston, the professor of modern history at St Andrews, and the architect Simon Molesworth, who sadly died of cancer a few years ago. Through Simon I met the love of my life, Hester Robinson, a Cambridge-trained architect. We got married in 1984 when I was a postdoc in London.

The Peterhouse cellars were kept up to scratch by the wine steward, Dr Adrian Matthias, a brilliant young mathematician who teased me about my ribosome research by asking: ‘Do I have ribosomes in my fingernails?’ I was not quick enough to answer: ‘No, but they make your fingernails.’ Dr Matthias was the man behind the Peterhouse Wine Society, which was officially run by Mark Willingale, another architect graduate friend. Mark surreptitiously introduced me to the Society, which was primarily intended for undergraduates. After all, a Cambridge graduate and real gentleman had to be able to tell a mediocre claret (the English name for red wine from Bordeaux, as I had learned by now) from a first-rate burgundy later on in life.

The Wine Society met about once a month in the most wonderful 15th-century rooms in Old Court, with sofas and armchairs and an enormous fireplace. The meetings were attended by a number of strikingly good-looking young men, no doubt the protégés of Dr Matthias. He certainly used his contacts in the top London wine merchants to present the best that vineyards in France, Italy, Spain and the New World could produce (even Germany, although it was almost impossible to buy a decent bottle of German wine in the high street). At one unforgettable meeting, Simon Loftus, who had inherited the Adnam’s brewery in Southwold (producer of the best real ale around Cambridge) and had become one of the premier wine sellers in the country, presented four or five consecutive vintages of Chateau Margaux, carefully explaining their relative merits and shortcomings to a clutch of beady-eyed twenty-year-olds. I have never had wine like this since.

But this was not really what I had come to Cambridge for. College life, the college itself, indeed the whole city of Cambridge, its ancient university, the other ancient and not so ancient colleges with their marvellous architecture, their gardens and romantic settings by the river, punting trips to Grantchester, concerts, feasts, May Balls – all these were bonuses, all the more special for being utterly unexpected. No German university, least of all Berlin, had anything comparable to offer. But the most special thing about Cambridge was its science, and the unique research environment. The MRC-LMB (for short: the lab) was the cradle of structural biology, actually the whole new science of molecular biology. That much I had found out as a year-out student at King’s College London. But now that I had landed in the middle of it, the reality surpassed all expectations. It took me some time to realise how lucky I had been; in fact, I did not fully realise it until some time after I left.

LIZARDS AT THE LAB

The young man Max had in mind for me as a PhD supervisor was Nigel Unwin. Nigel was in his mid-thirties, and looked more like one of the Beatles than anyone I had seen before or since – he still does. He was tall and lean, and greeted me with kind smiling eyes from under his fringe when Max’s secretary took me to meet him at his laboratory bench on the ground floor. In the months and years to come I was never quite sure that Nigel had really wanted a PhD student. Or rather, I was fairly sure that he had not really wanted one, and I do not know by what powers of persuasion Max had induced him to take me on. Nigel was one of the most intuitively brilliant scientists I would ever meet, but he evidently preferred working on his own. His most exceptional papers on the structure and mechanism of the nicotinic acetylcholine receptor he produced later on were single-author papers. But anyway, there I was, the first PhD student of Nigel Unwin – and almost also his last; I am aware of very few others, most notably Ron Milligan. I had been really lucky.

My project was to study the structure of eukaryotic ribosomes by electron crystallography. Two years before, Nigel and Richard Henderson, David Blow’s former student, had published the 7Å structure of bacteriorhodopsin determined by electron microscopy of unstained two-dimensional crystals called purple membrane. This was the work that Max had told me about on our frosty Dahlem walk. It had been a major breakthrough in several respects: it was the first time the structure of a protein – indeed any biological molecule – had been resolved by electron microscopy. It provided the first insight into the internal structure of a membrane protein. And it ushered in a new era in biological electron microscopy – a breathtaking development that continues unabated today. Membrane proteins and studying them by electron microscopy became the main theme of my own scientific life.

After their bacteriorhodopsin coup Richard and Nigel went in their own directions to lay the foundations of biological electron microscopy. Richard continued to investigate purple membrane, developing methods and writing programs for electron crystallography that enabled him fifteen years later to determine the structure of bacteriorhodopsin at near-atomic resolution – the first and still one of the best protein structures solved by the method. Nigel looked for other biological systems that would lend themselves to this marvellous new approach. In his quest he came across the work of an Italian zoologist, Carlo Taddei, who had discovered crystalline arrays of ribosomes in the unfertilised egg cells of hibernating lizards that inhabited the rocks and Roman ruins around Naples. Crates with a couple of hundred of these lizards, caught especially for Nigel, arrived at the lab from time to time and were put in the cold room to induce hibernation. One of the first things I had to learn in the lab was to dissect these lizards, take out the oocytes and prepare the crystalline ribosome patches for electron microscopy. For this, the poor dopey creatures were first decapitated. Nigel used a pair of surgical scissors and the tiny heads flew in all directions into inaccessible corners of the lab, where they rotted. Consequently, there was always a slightly putrid smell about Nigel’s laboratory. It did not seem to bother him. Ron Milligan later conjectured that Nigel had inadvertently cross-linked all his odorant receptors with glutaraldehyde and had no sense of smell or taste at all. I do not know how much of this is true, although the preparation of the ribosome crystals did involve a glutaraldehyde fixation step. Indeed I have not been entirely sure of my own sense of smell ever since, although it is still just about good enough to tell a poor claret from a good burgundy.

Nigel actually tried to establish a colony of these South Italian lizards on the south-facing side of the MRC building, where he had set a number of them free. Throughout my time in Cambridge, they could be seen on warm summer days basking in the sun on some small rocks that he had put out for their benefit. They were still there two or three years ago.

Figure 35.2. South Italian LMB lizard.

If egg cells of hibernating lizards harboured crystalline ribosomes, what other marvels of ribosome structure, crystalline or otherwise, might be hiding in the hundreds and thousands of species in the world around us? We briefly considered crocodiles as the nearest large relatives of lizards that might yield correspondingly large ribosome crystals, but the idea of keeping crocodiles in the cold room and dissecting them did not appeal. Nigel had an atlas of cell biology with full-page electron micrographs of the sub-cellular anatomy of various creatures. One of the most striking images was of a thin section of a bat pancreas, which showed concentric swirls of endoplasmic reticulum, densely studded with ribosomes. Didn’t bats occur in large numbers at nearby locations, for example Ely Cathedral? And didn’t bats hibernate? Might this not cause the ribosomes to crystallise on the ER membrane? Nigel wrote a letter to the Dean of Ely Cathedral asking for permission to capture a few bats for scientific experiments. Permission was granted by the verger, and one fine summer evening Nigel and I set out in his dark blue MG two-seater on the road towards Ely, Nigel at the wheel and me in the passenger seat, clutching a large net on a fifteen-foot pole. Nigel thought that the bats might mistake the fine-meshed net for a swarm of flies, which would attract them. That was the theory.

The sun was about to set when we arrived at the cathedral. We parked the MG and strode towards the great gothic edifice, net in hand. There were quite a few bats in the clear evening sky above. Nigel waved the net at them enticingly, but they were smart enough to stay well out of reach. The fly swarm theory did not seem to work. Nigel then asked me to have a go. I dragged the net through the air in vigorous figures of eight until my arms began to hurt, but to no avail. I had never felt so hopeless. Soon it became too dark to continue our exploits, and with a sense of deflation – in my case mixed with relief at not having to dissect any of these wonderful furry creatures – we took the road back to Cambridge.

Nigel taught me a great deal more aside from decapitating lizards. Virtually everything I know about practical electron microscopy I learnt from him. He was meticulous about aligning the microscope column, which in those days involved shifting the heavy magnetic lenses mechanically with a set of Allen keys. He taught me how to prepare carbon film and how to achieve perfect negative stain with uranyl acetate. His method of supervision was by example and by leading from the front. A few months into the project we talked about trying out a new contrasting agent for the ribosome crystals. Did I think that was a good idea? Yes, I thought it was a great idea. Would I like to try it? Yes, indeed I would. Nothing more was said, but when I had not done the experiment two weeks later, he went and did it himself, without further comment. This only happened once.

Other learning experiences included one when Ken Taylor, who together with Bob Glaeser had developed the first electron cryo-microscope at Berkeley and was now a postdoc at the LMB, stormed into Nigel’s lab white with rage. He had wasted an entire day, possibly more, by recording images of his actin filaments on film that had been loaded into the camera the wrong way up, so they all came out blank. He demanded to know which idiot had done this. It turned out to be me. Nigel smiled sternly. Again, this only happened to me once.

On another occasion, while examining my negatives at the optical bench I inadvertently undid the micrometer mechanism that centred the laser pinhole. It came apart completely, and all its tiny screws, springs and ball bearings scattered all over the floor. In dismay I confessed my misfortune to Nigel and was about to go to the workshop to ask them to fix it. But no, Nigel said I had to fix it myself. It took me two whole days, but I managed. I have been much more careful with delicate equipment ever since.

The optical bench was used to select the best electron micrographs of two-dimensional crystals for computer processing. Today, the processing of large image areas is commonplace and achieved in seconds with any personal computer or laptop. But in the early days of crystallographic processing even the Fourier transform of a 512 × 512 pixel array was a major undertaking. I still remember the sense of excitement when, about half way through my thesis, 1024 × 1024 arrays became possible – but each transform took two hours even with the LMB ‘mainframe’ computer, which was one of the most powerful ones available. Visualising the Fourier transforms for evaluation was another matter, because graphical displays of any sort had not yet been developed. Instead, the raw line printer output was laid out on the laboratory floor where it covered an area of 1.5 by 3 m, and had to be inspected from a suitable vantage point, standing on a chair or on the lab bench, to spot the reflections on the reciprocal lattice. Fortunately by the end of my PhD more convenient display options had become available.

Nigel was never a paragon of sartorial elegance. He dressed as if he was the inventor of ‘shabby chic’ long before it became fashionable. Well-worn (not actually torn) jeans, baggy grey jumpers with a few holes, unironed long-sleeved shirts partly stuffed into his jeans, and gym shoes with untied laces were his hallmark. As a scientist he had more important things to consider than his external appearance. There was however one occasion, years later, where this casual outfit just would not do. This was the Aminoff Prize ceremony in Stockholm. The Royal Swedish Academy of Sciences had awarded the Prize to Nigel and Richard for their groundbreaking work on purple membrane. They had very kindly invited me and Marin van Heel, the single-particle processing pioneer, to share the occasion as their guests. The Aminoff Prize stipulated very formal dress, presumably in preparation for other prizes awarded by the Swedish Academy. All four of us had to hire ‘white tie’, which consisted of black tails and trousers with cummerbund, white dress shirt and bow tie (for me this was the second and probably last occasion of wearing this attire, the other one having been the Grafton Breakfast at Peterhouse). Passers-by in the sober Stockholm street leading from the gentleman’s outfitters to the Swedish Academy must have been amazed to see four grown men dressed up like penguins (see Figure 35.4, p. 303), fooling around in broad daylight, taking numerous photographs of each other and having the time of their lives. But perhaps they were not amazed at all – perhaps they were used to it.

The LMB was well known for its Nobel Prize-winners, of whom it sported a higher density than probably any other institute in the world, as beautifully described in John Finch’s book A Nobel Fellow on Every Floor. On my first floor there was at the time only one: Max Perutz. John Kendrew had left to set up EMBO and the EMBL in Heidelberg. Aaron Klug was awarded the Prize in 1982, the year after I left. An apocryphal story has it that when the press wanted to take a photograph of Aaron next to the electron microscope that had produced some of the Prize-winning results, he had to be shown where it was. Either he had forgotten or, possibly, he had never used it himself. Fred Sanger, who counted double because he had two Nobel Prizes, was on a different floor. I frequently saw him at the back of the elevator up to the canteen, where he looked as if he had just come in from doing the garden, smiling benignly. The notorious Jim Watson, who had gone back to the US years earlier, I only met at the Max Perutz memorial symposium at Robinson College in 2002. He shook my hand firmly, looked me straight in the eye, and said ‘Watson!’, as if anyone would not recognise him. Francis Crick had left for the Salk Institute shortly before I joined Nigel. I met him at a fairly wild party thrown by Nigel and his charming wife Janet at their house in Linton, just south of Cambridge. Nigel knew Crick well and took over their house, the ‘Golden Helix’ in the city, when the Cricks moved to California. Janet and Nigel still live there. To the party in Linton I had taken Sylvie, my then girlfriend, a petite, very pretty French language student. Nigel approved totally. I do not think there was anything he approved of more during my entire PhD. For months afterwards he asked, ‘And how is Sylvie?’ I should say that Sylvie was no less taken with Nigel. She kept telling me what a nice man she thought he was. He certainly was, but it seemed slightly unnecessary to remind me of it quite so often.

PhD STUDENT IN THE DIVISION

Being a PhD student in the Structural Studies Division, there was nothing one could not ask, and no question was left unanswered. If Nigel was busy at the electron microscope, as he usually was, I only needed to walk down the hall. In the short distance to Max’s office before the staircase, with its great model of tobacco mosaic virus made of polystyrene for the protein and red garden hose for the RNA, I passed the offices of Aaron Klug, Hugh Huxley (the joint Heads of Division), Richard Henderson, Linda Amos, John Finch and Joyce Baldwin, all of them pioneers of biological electron microscopy. Turning the other way, there were Bob Diamond, Andrew McLachlan and Tony Crowther, mathematicians turned crystallographers or electron microscopists who knew, and were able to explain, everything about Fourier transforms. Their doors were always open (one of Max’s first deeds as the LMB director was to have all the locks removed), and it was possible, even encouraged, to wander in and ask a question. There was no need for formal supervision; I picked up what I did by osmosis in this tightly packed, uniquely rich environment.

Apart from this exceptional corona of structural biologists, there were postdocs, mainly from the leading US labs. In the next office that could be reached only by squeezing past my desk were Schutt and Gerard Bricogne. Schutt, who seemed not to have a first name (I later discovered that it was Clarence), sometimes stopped to quiz me about my project and molecular biology in general, exposing gaping holes in my knowledge. ‘Werner, you must study!’ he would admonish me. Gerard, a dapper, well-dressed Frenchman with a beautiful girlfriend, had already achieved some notoriety in crystallography circles with his program called ‘The Joy of Skewing’. On one memorable occasion he gathered the six or seven brightest minds in the Division into a small seminar room to describe his maximum likelihood method for the first time. They came out three hours later looking pale, exhausted and nonplussed. Those I asked afterwards thought that possibly Aaron Klug and Tony Crowther might have got something out of Gerard’s expostulations of this non-intuitive approach that later revolutionised structural biology, but they were unable to explain it to me.

There were countless visitors; the LMB was the Mecca of structural biology. Bob Glaeser, Michael Rossmann, Joachim Frank, Tom Steitz and later Wah Chiu came for sabbaticals; Jim Lake gave Nigel one of his colourful ribosome models (Nigel told me later that his daughter Phoebe liked to play with it). All the great and the good of this still young field came through, gave talks, discussed or did experiments. In retrospect it strikes me that there were hardly any other PhD students. PhD students only got in the way and interfered with serious work, seemed to be the lab philosophy. I was certainly a case in point, as my optical bench and film loading episodes had shown.

Of my contemporaries, only Jonathan Scholey, the ace soccer player who was with Jake Kendrick-Jones and is now professor of cell biology at UC Davis, and Steve Fuller come to mind. Steve was there for a year on secondment from Rod Capaldi at the University of Oregon, to work on two-dimensional crystals of cytochrome oxidase with Richard. Steve, who was a couple of years younger than me, is the only person with whom I tried my hand at tennis. We played a total of three games at the recreational facilities of Addenbrooke’s Hospital, the Frank Lee Centre, before we both decided over a pint at the bar that whatever talents we had, playing tennis was not one of them – a wise decision. Steve later became my de facto boss as the joint coordinator of the Structural Biology Programme at EMBL Heidelberg, during my time there as a group leader – an uneasy relationship, not free from rivalry. When he subsequently took up a Wellcome Principal Investigatorship at Oxford he tragically developed severe multiple sclerosis. He is now in a wheelchair under constant care with his family in Oregon – too far for me to tell him in person that I regard him as one of the true pioneers of electron cryo-microscopy, and what a great job he did as Programme Coordinator at the EMBL.

Some time during my second year Nigel announced that he had accepted a professorship at Stanford University and would move there within a few months. He would take with him as a PhD student Ron Milligan, who had recently joined him as a laboratory technician from London Zoo. There was no mention of me. Nigel thought it was better if I finished my PhD at Cambridge, and he was right. I was well along with my project, able to work quite independently, and he had always been a hands-off supervisor of the LMB ‘sink or swim’ persuasion. Richard became my de facto supervisor and I did not sink. My contact with Nigel at Stanford was by (regular) mail. When I sent him the first draft of my thesis, he asked Ron Milligan what he should do with it. ‘Read it!’ was Ron’s advice, as he later told me.

POSTDOC AT THE ETH

The years at the LMB were the most formative period of my life. Apart from teaching me a new experimental technique – electron crystallography – and kindling my future research interest – membrane proteins – it also provided me with a set of standards, values and rules to live by as a scientist. This was not always to my advantage: these rules and values were not shared universally, as I soon found out.

I had decided to go to the ETH Zürich for a postdoc to work on the plant light-harvesting complex LHC-II, one of the few membrane proteins that form almost perfect two-dimensional crystals, as I was able to show later. Richard was highly sceptical – LMB PhDs went to places like Harvard, Yale or Stanford for their postdocs. The ETH Zürich – even then one of the best universities in continental Europe – did not feature on his radar. ‘If you go there, you will never be seen or heard of again’ were his words. Events very nearly proved him right, but he could not have known how. It was the only time I disregarded his advice.

At the ETH I joined the group of Kurt Mühlethaler in the Institute of Cell Biology. Moor and Mühlethaler had developed freeze-fracture EM as an important new technique for the study of biological membranes in the 1960s and 70s. Both were now professors, but they had not kept up with the exciting recent developments in biological EM. Mühlethaler was only a few years from retirement and spent much of his time cutting his garden hedge, as he would tell us over coffee. His assistant Ernst Wehrli had a keen interest in photosynthesis research and had observed that LHC-II, this most abundant of membrane proteins, formed crystalline aggregates upon salt precipitation. The freeze-fracture micrographs of these arrays had attracted me to his laboratory in the first place. Wehrli showed me how to isolate LHC-II from chloroplasts (amazingly easy, because there was so much of it), and I developed a protocol to grow usable two-dimensional crystals. I recorded electron micrographs at various tilt angles, processed them and worked out a first three-dimensional map, all entirely by myself. I then wrote a manuscript and sent it to Nature, not before having shown it to Mühlethaler. He did not request to be an author, and after my time at the LMB it did not occur to me that he might want to be. I should have known this was a mistake. The unwritten rules of Teutonic academia (which definitely encompassed the ETH) entitled professors to authorship on papers to which they had not contributed and knew little about. Only recently has this practice been branded as unethical in Germany, largely because of students, postdocs and group leaders joining universities and institutes from the UK, the US or the EMBL, where independence in young scientists is valued and encouraged. When my manuscript was accepted, Mühlethaler was at first delighted, but when a few friendly colleagues took him to one side and asked why he was not an author on the paper, I was promptly and unceremoniously sacked.

In the end, my sacking from the ETH was a blessing in disguise (Matti Saraste, my fellow group leader at the EMBL, once kindly remarked that it was the one thing I had in common with Albert Einstein). With a hastily arranged long-term EMBO fellowship I took refuge in David Blow’s and Jim Barber’s labs at Imperial College London, where I was able to continue my work on LHC-II unencumbered by the Mühlethalers of this world. For some time I commuted to the LMB for two days a week, where Richard helped me to get to grips with a tricky problem related to disorder in the multi-layered thin crystals I had grown. Having struggled for some time I wrote several options on a piece of paper, which I left on his desk before taking the train back to London one night. One option was to give up. ‘Never!’ Richard scribbled on the note, which I still have (see Figure 35.3). This time I heeded his advice, and I am very glad I did. In due course I was able to determine the structure of LHC-II at near-atomic resolution by electron microscopy, inspired by Nigel’s beautiful cryo-EM studies of the acetylcholine receptor and following in the footsteps of Richard’s high-resolution bacteriorhodopsin work. I owe both of them, Max and the whole LMB more than I can say.

Figure 35.3. Note left by the author on Richard Henderson’s desk.

Figure 35.4. Michael Rossmann, Werner Kühlbrandt, Nigel Unwin and Richard Henderson, in Stockholm, Sweden. (See p. 299.)

CHAPTER 36

My Days with Aaron at the MRC-LMB, January–May 1979

Stephen J. Lippard

Department of Chemistry, MIT, Cambridge, MA

Figure 36.1. Aaron Klug, Bob Diamond and Steve Lippard at a meeting in honour of Aaron Klug, 9th September 1996, Peterhouse, Cambridge.

I have had six sabbatical leaves in my career, but none quite as memorable as the one in Cambridge in the winter/spring of 1979. Trained at MIT as a pure inorganic chemist in the laboratory of F. Albert Cotton, I investigated the chemistry of the bromorhenates for my PhD degree. But my passion, then as now, has always been at the interface of inorganic chemistry and biology/medicine, so sabbaticals were essential for me to help convert ideas into reality by learning the methodologies necessary to direct the work. My Cambridge sabbatical came at a time when my lab began to look seriously into the molecular mechanism of the anticancer drug cisplatin, which binds DNA as its principal cellular target. It seemed to me that knowledge of platinum binding to nucleosomes might be important and, in part because I had always wanted to spend some time in England, I sought out Aaron Klug and secured a post in his lab for five months, from January to May 1979. I had no idea at the time what a wonderful choice that would turn out to be.

From a previous sabbatical experience I came well prepared for Cambridge. I knew that it was important to arrive with a well-defined project in mind. I decided to address the nature of platinum anticancer drugs binding to nucleosome core particles, and the Klug lab was the perfect place to undertake such an endeavour. I had enthusiastic encouragement and kind hospitality from Aaron as well as wonderful experimental help from, especially, Len Lutter, Barbara Rushton and Jo Butler. Len was a terrific daily mentor. Among other things, he taught me how to run gels, set up radioactivity assays, and analyse proteins, DNA, and platinum in a single experiment! He was a gifted and patient mentor. I had 195mPt-labeled cisplatin, 195mPt being a gamma emitter with a four-day half-life, shipped from Oak Ridge National Laboratory in lead containers through the regular mail (imagine!) by my collaborator Jim Hoeschele, who was working at Oak Ridge at the time. Barbara supplied me with core particles, and Jo helped with sedimentation velocity analyses. The studies were most successful and led to a first-author PNAS paper in December 1979, which really impressed my research group back at Columbia University in New York where I was then a professor. Each day was for me a joy, not only because of the science, but for everything else that the MRC and Cambridge provided.

Aaron helped arrange for housing in Churchill College, which had flats for Americans. Churchill was surrounded mainly by rugby fields, which provided a much more pastoral setting than today. Aaron belonged to Peterhouse, so being a fellow of Churchill was not an option, but that hardly mattered for I came in January with my wife Judy and two sons Josh and Alex, ages ten and four-and-a-half respectively, and spent most of my non-working time with them. The boys were enrolled in the Newnham Croft School in Cambridge, which was wonderful, possibly the best educational institution they ever attended. Alex picked up a perfect English accent, and when we returned to the States he came home from school with tears to tell me: ‘Daddy – I can’t speak American any more!’ That lasted two days. We bought a used Renault with floor stick shift (you could see through that floor) that sometimes defeated Judy but did the job, and I hired out a moped to take me the five miles to the MRC and then back each day. Because of my busy schedule, that moped was critical.

These were the days before the Internet of course, which worked greatly to my advantage. I spent every day except Thursday at the MRC, but had all mail and correspondence (except my platinum samples) sent to the Inorganic Chemistry Department on Lensfield Road where Jack Lewis (now Lord Lewis) kindly provided me with an office. As an inorganic chemist, I was interested to meet the visiting speakers, and Thursday was inorganic seminar day at Lensfield Road. There were great pubs in that area as well, which Jack and Brian Johnson introduced me to on many occasions. My usual was a ploughman’s and a pint. On the way back from Aaron’s lab I would stop each evening to pick up my mail before heading home to Churchill. It was a great way to keep focused on my research at the LMB without the many unwanted interruptions that email can now demand if you let it. The flats were comfortable enough, but on occasion I had to brush off a very light dusting of snow that had seeped through the not-too-tight windows to tuck in the boys as they were falling asleep.

Science at the lab was booming. Aaron was just about the smartest person I had ever met and we had a great time discussing science. I love his Dickensian name (Klug), which I later could compare to that of my MIT (I moved there in 1983) colleague Phil Sharp – both giant intellects. At the time, Aaron was engaged in many activities, one of which was soon to become one of his great discoveries, the ‘zinc finger’. That work probably did more to expose the importance of inorganic chemistry to biologists than any other single achievement. Now even geneticists would realise that metals in biology were more than an idle curiosity. His lab was also working hard to determine an X-ray structure of the nucleosome core particle, and I was happy to contribute in a small way to that ultimate achievement. I had carried with me to Cambridge some multi-heavy-atom inorganic compounds, including TAMM and PIP, which were later used by Aaron and Tim Richmond, then a graduate student of Aaron, to solve the structure. TAMM, for tetrakis(acetoximercuri)methane, had been used in my lab in early studies aimed at sequencing DNA in the electron microscope. The idea was to attach the label to specific nucleobases and then read the heavy atom positions along the DNA in a scanning transmission electron microscope (STEM). Our trick was to label the ‘soft’ 5′-phosphorothioate sulphur atoms appended to the bases with a heavy atom such as mercury or platinum or a multi-heavy atom cluster. With a polymerase we could replicate DNA with deoxynucleoside triphosphates α-S. Thus every dG, for example, would have a flanking sulphur-atom and in principle one could get the sequence with just two labelled DNAs, one with A and G and the other with C and G, getting T from the absences. With important help from Fritz Eckstein in Germany who supplied the sulphur-modified dNTPs, we were able to manage quite specific labelling, but the STEM could not handle the samples without motion that obscured the desired information. PIP was a side-product made by my graduate student Tom O’Halloran during his studies of platinum blues and related compounds – it contained two platinum atoms bridged by two iodide ions. Both TAMM and PIP provided plenty of electron density to help phase the core particle crystals.

Lunches and coffee breaks in the canteen on the top floor of the LMB offered a wonderful opportunity to meet and discuss science with great people. I had a special fondness for the late Max Perutz. Max loved to talk about haemoglobin with me and we spent hours discussing aspects of its structure, dioxygen binding, and other properties. Richard Henderson and Daniela Rhodes were special people and I greatly enjoyed learning about their science. Mike Gait taught me a lot about methodologies for handling nucleic acids. I had only casual conversations with Sydney Brenner, Hugh Huxley and Fred Sanger, but their groups were a great source of inspiration. I had my first lessons in macromolecular crystallography from John Finch in addition to Aaron. I think by the end of my five months I finally understood something about image reconstruction, although as a small molecule crystallographer I did not find it as compelling as ordinary small molecule X-ray crystallography. Luckily, the Nobel committee appreciated the value of three-dimensional image reconstruction and the beautiful structures it could produce, and in 1982 Aaron was awarded the prize for his pioneering work in that area. At the LMB I learned enough about DNA sequencing to be able, on my return to Columbia, to inspire my postdoc Tom Tullius to get it set up in my own lab in the chemistry department. I marvel now as then that our inorganic chemistry lab at Columbia was the first to do DNA sequencing, I believe, and people came to Tom to learn how to run the gels.

There was a tremendous heterogeneity in the MRC Laboratory of Molecular Biology – many different kinds of science were being conducted, and there never seemed to be enough space for all the people who were crowded in there. I later came to realise that the crowding of people in a lab environment can be a good thing, as long as safety is maintained, for it breeds cross-fertilisation of ideas. I recall at Easter the delivery of a couple of crates of baby chicks to my lab, thinking how nice that someone was going to take them home to their kids for the holiday. They made a terrific racket, but as I was working up some data at my desk I noticed that the noise was slowly subsiding. When I looked around and saw a lab technician extracting tubulin (if I recall correctly) from their brains – I decided that I could take off a little early that afternoon.

By the end of May, I had discovered that cisplatin bound largely to DNA in nucleosomes, produced DNA-histone cross-links, and cross-linked the histones. Control experiments with the biologically inactive trans isomer of cisplatin produced a greater proportion of cross-links, and others eventually used trans-DDP as a general cross-linking agent. Twenty-five years later, Matthias Ober, a postdoc in my lab at MIT, did some terrific quantitative hydroxyl radical footprinting of nucleosomes and learned that site-specific cisplatin adducts can override the natural positioning sequences of DNA on the core particle. My graduate student Ryan Todd subsequently managed to obtain a medium resolution crystal structure of a site-specifically platinated core particle, which confirmed and extended the findings of Matthias. I don’t today think that the anticancer activity of cisplatin has much to do with its binding to DNA on the nucleosome, but nature is full of surprises and I keep an open mind.

The lab work that I did in Aaron’s group was the last serious science I performed with my own hands – something that I sometimes regret. My MIT colleague, the late Gobind Khorana, served as a good role model; he demonstrated that it is possible to keep active at the bench throughout one’s career. Although bioinorganic and medicinal inorganic chemistry has changed tremendously in the intervening years since MRC, the ability of biologists, biochemists, molecular biologists, cell biologists and medical scientists to appreciate the importance of inorganic elements to their fields has not materially changed. Most still do not think of a ligand as something that binds a metal ion. I find this situation somewhat amazing. More than half of the people treated with drugs for solid tumours get a platinum compound today, yet many biological scientists could not write the chemical structure of cisplatin or most other inorganic coordination compounds if pressed. The interface between inorganic chemistry and neuroscience is an area that is especially ripe for study, and I hope that young scientists who might be reading this little essay might be inspired to try their hand at it.

What I learned from my sabbaticals, and especially the one with Aaron, is that the people you meet who come at science from a different perspective are most often the ones from whom you can derive the greatest inspiration. To be sure, the LMB was then and hopefully remains a very special place. There were no barriers, it seemed, to collaborations; the physical layout of the building and especially the canteen provided great thought incubation chambers, the presence of so many brilliant individuals, and the simplicity of life compared to the present – with the incessant bombardment from the Internet and all that is associated with it – were most conducive to creative thinking. And beautiful Cambridge, unencumbered by commerce breeding on science, ready to accept the lone scholar, offering a quiet place to think and to work and to walk about, was an important part of the whole experience. The only thing that was missing, in hindsight, was a cadre of senior women scientists – I hope that the situation has changed today.

In closing, I wish to thank Hugh Huxley for inspiring me to write this little essay, which perhaps someone will find interesting. I take full responsibility for its inaccuracies, for memory (zinc in the hippocampus plays an important part) is a strange human frailty as I come to realise more with time. History is the survival of the best-told tales, I have long believed.

CHAPTER 37

LMB Memories from 1970 and 1979

Donald Engelman

Department of Molecular Biophysics and Biochemistry, Yale University

I first met the LMB briefly, when I came up from London to give a talk in 1970. I was working with Maurice Wilkins, applying X-ray scattering to study membrane structure. The work was going well, giving a proof of lipid bilayer structure and documenting a lipid phase transition, and I presented it to a small audience, who asked sharp questions; then I was given five pounds for expenses and got back on the train to London. Not a very deep experience, but I found the atmosphere stimulating, and when I later had some sabbatical time I wrote to Richard Henderson, whom I knew from Yale, to ask about a visit. He had me write to Aaron Klug, and it was agreed that I would come.

I was there with my family, and although I had a visiting appointment at Trinity College it rapidly became apparent that I could not participate much – as Richard pointed out to me: ‘There are three things in Cambridge: lab life, family life, and college life, and you can only ever do two of them.’ So I settled in, sharing an office with Clarence Schutt and Phil Evans, and chatting with Richard. We had some adventures, including a kayak trip with Richard down the Cam, and learning the hard way that you should not try to pull your punt pole if it sticks in the mud: I believe my plunge into the water was documented by a group of Japanese tourists who gleefully photographed it from a nearby bridge.

One topic that captured us came from Yuri Ovchinnikov, who was running his lab of over 100 people to determine the protein sequence of bacteriorhodopsin, and we had heard he had completed the sequence, which contained seven hydrophobic stretches of presumed transmembrane helices. Richard and Nigel Unwin had published (in 1975) a 3D map with seven sausage-shaped densities that showed, for the first time, the transmembrane helix structures that have guided thinking in membrane structure ever since. We discussed the task of how to assign the hydrophobic sequences to the seven densities, a problem that had seven! or 5,040 possible solutions.

The first problem was to obtain the sequence, with Ovchinnikov showing it in his talks but moving on to the next slide before anyone had time to write it down. The solution we came up with was to go to a talk he was giving (in London, as we recall); I would then start copying it down starting at the N-terminus and Richard would do the same starting at the C-terminus. We had less than a minute to copy over 120 amino acids each, but by the time he moved to the next slide, we had managed to get an overlap. After his talk, we showed him the sequence to confirm it, and then he gave us a copy. We then spent a month or two (with Andrew McLachlan and Bonnie Wallace) trying to come up with credible arguments to narrow down the number of possibilities to one, including integrating the densities in the EM projection map and imposing length constraints from the loops between helices. We had a sweepstake, but everyone seemed to put money on the same (subsequently shown to be the correct) model. We decided we could only publish a paper if we came down in favour of a single model, but as a caveat we had a table of 41 possible models (based on numerous criteria) with the top model being our favourite. In due course, ten years later, it was shown to be the correct one, but not before Agard and Stroud had tried something similar and come out in favour of our number two model (maybe they felt the need to be different). We greatly enjoyed the adventure, with Brian Matthews even kindly referring to it as having needed the same kind of hand-waving model-building as the DNA double helix. About a year later, Khorana found two adjacent tryptophan residues where the Moscow group with Abdulaev and Ochinnikov had only one, so their sequence was almost perfect.

Thinking about this problem led me to try using neutron scattering to test the model, by supplying specific deuterated amino acids in the growth medium of the Halobacterium, and using difference maps to find them. This work took a while, but eventually gave some supporting evidence. I remember asking Sydney Brenner, then the head of the lab, for funds to buy the deuterated amino acids, and he initially didn’t want to pay for them, but eventually he did.

Later, partly as a result of the work that started in the LMB, Richard and I both went to a retinal protein meeting at Lake Baikal, near Irkutsk, that Ovchinnikov organised, but could not attend himself because by then he had contracted leukaemia. Richard took a great photo of me standing in front of a Baikal poster and another of a heroic Russian worker, angrily advocating peace, that encapsulates our bacteriorhodopsin adventure.

Figure 37.1. Lake Baikal poster.

CHAPTER 38

Two and a Half Years at the LMB That Imprinted My Scientific Career (1980–83)

Raúl Padrón

Center of Structural Biology, IVIC, Caracas, Venezuela

In 1966, while still attending high school in Caracas, I joined Dr Karl Gaede’s lab at the Biochemistry Department of the Venezuelan Institute for Scientific Research (IVIC). In his lab I was engaged in building a 2D-electrophoresis setup. One year later I started studying electrical engineering at the Central University of Venezuela, which was then closed for the next two years. In the meantime, I moved to the Biophysics and Biochemistry Centre joining first Dr Jorge Villegas’ lab – while Dr John Nicholls visited him to work on recording signals from the neurons in the leech ganglia – and then to Dr Carlo Caputo’s lab with the aim of understanding how the giant muscle fibres of barnacle were activated by using a calcium dye indicator. Once graduated I joined the lab of Dr Leonardo Mateu to study the structure and function of the nerve myelin sheath by low-angle X-ray diffraction and electron microscopy. I received my MSc in 1977 and my PhD in physiology and biophysics in February 1980, and joined IVIC as Associated Investigator.

While I was still wondering about a place for a postdoctoral stay, Dr Vittorio Luzzati (Dr Mateu’s supervisor) visited the lab and suggested that I should write to Dr Aaron Klug at the MRC-LMB. He accepted me to work on a high resolution 3D-map of tobacco mosaic virus, starting in September 1980. IVIC awarded me the Vollmer Excellence Fellowship to support my stay in England. In the next few weeks I read several structural papers. One (Journal of Molecular Biology, 1967. 30, 383–434) captivated my attention and its author (Dr Hugh E. Huxley) happened to be at LMB. I realised after reading that paper how fascinating was the structure and function of muscle which involved force and movement. At that time Dr Humberto Fernández-Morán visited the lab and he also mentioned Huxley’s recent exciting synchrotron research, and suggested that I should work with him. I did feel brave enough to write again to Dr Klug telling him my great interest in working – instead – on muscle contraction. Dr Klug agreed without hesitation and put me in contact with Dr Huxley who approved my postdoctoral stay at his lab. With this gear change, I concentrated on muscle structure papers, reading all of Huxley’s papers I could find at IVIC library; as well as attending an intensive English course. It was going to be my first trip (and flight) abroad. What awaited me on the other side of the Atlantic Ocean was not only a new country and people, but also an extraordinary cultural and scientific shock. Everything that happened after I landed in England was a surprise for me, including the adventure in muscle that was about to begin.

I arrived at Cambridge train station on 1 September 1980. I quickly learned that in this new country the way of life was very different: the first surprise was that people queued for a taxi. I had never in my life seen that. That was only the beginning of many cultural differences between Venezuela and England. Ms Jenny Brightwell kindly received me with the news that Dr Huxley was kind enough to let me stay in his house while he was away in Woods Hole. People at Cambridge were indeed very kind and helpful. I soon discovered that the English that I had learned in Caracas was not precisely British English, so for a few weeks I was slightly perplexed when talking to people. I went to Hills Road to visit the famous LMB without delay. I mentioned to someone in the building’s entrance that I was there to work with Dr Huxley, and asked where I could find Dr Klug. They told me where Aaron’s office was, joking that everybody was a doctor there. I introduced myself to Aaron, thanked him for the opportunity to work there and briefly mentioned the project I wanted to do with Hugh.

Somehow I found myself in room 213. There, Roger Craig, a postdoc from Australia, knew in advance that a new postdoc from Venezuela was arriving. Roger briefly showed me the lab, and in the next few weeks educated me in brief about it, LMB customs, the Cambridge way of life and the weather. Coming from a tropical country, and not knowing what to expect there, soon I started asking him: ‘Roger, is it the winter yet?’ when the temperature dropped to 15°C! Two more people joined the room: Marcus Kress, a student from Germany who came to do his PhD with Hugh, and later Sengen Xu, a postdoc from China. That completed the capacity of the room of four desks, with four different nationalities, continents and languages. In retrospect, sharing rooms was one of the successful features of the LMB. After less than a week, Aaron pointed out to me another important key feature, when he politely mentioned to me that he noticed I was missing the coffee and tea times by working in the lab instead, stating seriously that going to the canteen was indeed very important. In fact, after this advice I started going to the canteen more frequently and finally every day, discovering for myself that, with the nice excuse of drinking coffee and tea, LMB people have the opportunity twice a day to discuss very informally – sometimes between different tables – ideas, current experiment results, failures and many other topics.

Hugh returned some weeks later from Woods Hole. He had pioneered the muscle field by discovering, with the late Jean Hanson (Roger’s PhD supervisor), that the muscle sarcomere, the tiniest microscopic unit capable of shortening and producing mechanical force, was formed by two sets of filaments, the thick myosin-containing and the thin actin-containing filaments; he proposed that the myosin heads protrude from the backbone of the thick filament and cyclically interact with the thin filament, forming crossbridges and actively producing force while shortening the sarcomere. He also provided the first direct evidence for the tilting of these cross-bridges during force generation in muscle. We discussed the project I proposed to do in his lab – to use relaxed micro-dialysed single giant barnacle muscle fibres, studying them by X-ray diffraction to learn about their activation by calcium. As getting and keeping these bizarre animals could become a problem, Hugh suggested instead trying to ‘freeze’ biochemically the cross-bridge cycle by using AMPPNP, a non hydrolysable ATP analog. It sounded very interesting to me and I started reading and preparing to do the experiments (Figure 38.1, left) while Hugh showed me how to set up and align a Huxley-Holmes mirror-monochromator camera setup on the big wheel X-ray generator. It surprised me that muscle fibre X-ray diffraction patterns were recorded on film, as in Venezuela I had recorded the one-dimensional X-ray diffraction patterns from the myelin using an electronic linear position sensitive detector, which we learned to build while Andre Gabriel was visiting from the Luzzati lab. Learning directly from Hugh how to prepare glycerinated rabbit psoas muscle was indeed an interesting experience, especially the rabbit preparation and dissection. Luckily after a few weeks I managed to get some good rabbit psoas rigor diffraction patterns. I still remember clearly the day when I developed and fixed in the dark room my first muscle fibre X-ray diffraction film: the room was dark, and someone else was there; I asked this person to help me in developing my exposed film as it was my first one. This person kindly helped me, and when I asked for reassurance that he really knew what he was doing, the person replied that he knew quite well. Once the light was on I discovered the person was Max Perutz!

Another feature of LMB was the frequent visitors to the lab. One day Ken Holmes visited the lab and Hugh mentioned my recent rigor X-ray diffraction patterns. Ken was kind enough to assess the quality of my initial rigor patterns as very good, especially one, boosting me to keep working hard on it. In fact the AMPPNP project kept me very busy until the end of 1982 when I completed it. I accompanied Hugh, Wasi Faruqi, Marcus Kress and Bob Simmons on one of their visits to the DESY synchrotron in Hamburg. I clearly remember the moment in which a reflection started to change in the screen and Hugh’s excitement was so extreme that I worried he might have a heart attack. Wasi Faruqi helped me with setting and using his position sensitive detectors for recording the meridian and equatorial X-ray reflections of the AMPPNP-treated muscles. Tony Woollard was always there helping to maintain the big wheel generator I was using and ensuring that it continued to work well during the long exposure runs. Another lively LMB moment showed the importance of experimental data for a scientist; one morning there was a fire in the building, and Hugh literally jumped out with a stack of computer tapes, computer listings and films, refusing to risk losing them in the fire. Later I had the valuable opportunity to record some synchrotron data for my project in the Daresbury synchrotron (in July 1982), joining a trip there with Hugh and Marcus.

During my stay at LMB I had the opportunity to attend many meetings, including the MRC-LMB Laboratory Symposium, six meetings in the UK, the 1981 Muscle Gordon Research Conference and the 1983 Alpbach Workshop on Muscle. The Laboratory Symposium was especially important for me. On another occasion, by mistake, I attended a meeting that I later discovered was intended only for the LMB permanent staff. To my surprise, in the meeting room there was a panel of Nobel Prize-winners leading an open and free discussion on – from what I understood – the future of the different divisions, labs and units at LMB. Quite a learning experience about science planning and revision for a visitor from other, more tropical latitudes! The fact that Aaron and Hugh were joint heads of the LMB Structural Studies Division was an unimaginable concept where I came from. The LMB building was busy all day and all night, including of course the weekends; it was like a scientific factory that could not stop production, driven by curiosity. Hugh’s lab had periodic meetings of the Muscle and Cell Motility Discussion Group, known informally as the ‘Muscle Club’, to which I contributed a few times. Once, I told Roger that a speaker at these talks spoke very clear and understandable English for me. Roger explained why Murray Stewart’s English was so familiar to me: I had learnt and practised my spoken English mainly with Roger, another Australian, and Murray was the second Australian I had heard in my life! I finished the AMPPNP project by the end of 1982 and we wrote a paper that was published later, in 1984 (Journal of Muscle Research and Cell Motility 1984. 5, 613–55), the same year as a paper in collaboration with Ed Egelman – who was a postdoc in the office opposite – on the actin molecule disposition on the native thin filament (Nature 1984. 307, 56–8). I still remember when I was in Jenny Brightwell’s office, and a telex message came through announcing Aaron’s 1982 Nobel Prize in Chemistry. The news spread around the building in minutes and in a very short time Michael Fuller (another key LMB figure) organised a celebration party in the canteen. At that party I discovered that people in England queue not only for taxis, but also for champagne cups! I vaguely remember hearing Michael say that champagne was always prepared in advance for the next Nobel Prize-winner. In fact, while writing these recollections I heard on the radio that John Gurdon had won the 2012 Nobel Prize for Medicine, and right after I arrived at the LMB Fred Sanger won his second (1980) Nobel Prize for Chemistry (by the way, the same year as the Venezuelan Baruj Benacerraf won the Nobel Prize for Physiology or Medicine). Another feature of the LMB was the rapidity with which Michael provided any biochemical you needed for an experiment, i.e. no grant writing! I was not aware of the concept of writing grants until I had the great opportunity to help Roger to prepare the final version of an application he was writing on a typewriter, during a very long day and night that finished the next day. Once or twice I came close to staying forever in Cambridge, not working there but either frozen alive during the coldest winter in 50 years, or passing away after falling down when my bike slipped on ice – an unknown phenomenon for me – or a lorry smashed my bike on a roundabout, or drowning while punting on the Cam – fortunately quickly rescued by a young Briton. All of these memories would have been enough to fill the rest of the life of a young Venezuelan scientist, altering my whole future scientific career. However, another LMB feature changed it completely.

At the LMB we had complete intellectual freedom. Sharing the office with Roger proved to be an important turning point in my life as a scientist. Roger was working on viewing metal shadowed myosin molecules in the electron microscope to understand its function. We talked a lot every day about the experiments we were doing on our different projects, and many times were joined by Marcus with his results. We became great friends and spent a lot of time talking about muscle and many other things. We wondered if the structure of a thick filament – a big supramolecular arrangement – could ever be solved by structural techniques. Marcus was building a computer model of myosin heads on a thick filament using spheres to calculate its X-ray diffraction pattern. He wanted to use it to test John Haselgrove’s proposal that the myosin heads were pointing in opposite directions along the thick filament axis. Roger and I were discussing the possibility of solving the structure of a thick filament by negative staining – a technique developed by Hugh 26 years earlier. At that time Roger was collaborating with Peter Vibert at Brandeis University doing electron microscopy of scallop thick filaments.

Roger was very keen that I should attend different muscle meetings. He pointed out that a very important one was the Muscle Gordon Research Conference to be held in Holderness School, Plymouth, by the end of June 1981. I applied, waited for a while and was accepted right before the meeting; luckily I was able to attend. Roger was right, this meeting was superb scientific experience for me. I had the opportunity to meet and talk to many famous muscle scientists. One, John Wray, was doing X-ray diffraction patterns of muscles from a variety of invertebrates. He told me that according to his X-ray diffraction patterns, the thick filaments from tarantula muscle were particularly well helically ordered, and advised me to view them in the electron microscope. After coming back to Cambridge, John visited the lab and gave me two beautiful tarantula X-ray diffraction patterns. Roger and I decided to pursue the new project together to visualise by negative staining isolated thick filaments from tarantula muscle with the hope – if they were indeed so well ordered – that we could even calculate a 3D reconstruction. Soon we made a short trip to a nearby town to buy a big tarantula in a pet shop. We did try the muscles from this tarantula, making a leg muscle homogenate one weekend in October 1981, and discovered that their thick filaments were indeed beautifully ordered. After that initial success we kept working on that topic – as well as our main projects – for the rest of 1981 and 1982. Hugh liked this unexpected digression from our original plans and encouraged us in pursuing it. We succeeded in getting low electron dose pictures in the electron microscope. I still remember one day when Aaron brought some visitors to the biochemistry room where I was doing a dissection. He wanted to show them the electron microscopes. Brian Pope politely reminded him that the microscopes had been moved to another floor a few years before, something that Aaron acknowledged immediately as he had completely forgotten. We asked Tony Crowther for advice on image processing and after some time we started collaborating with him in learning how to calculate a 3D reconstruction from first principles. The first 3D map was obtained in March 1982. It is worth mentioning that the 3D reconstructions then were calculated in the computer room using the VAX-11/780 computer and that in 1982 the computer displays were only monochrome text monitors. Claudio Villa made some balsa wood stack models for visualising the 3D reconstruction and Tony taught me how to prepare a perspex stack 3D map. Ed wrote a clever computer program that enabled the surface representation of the tarantula thick filament to be displayed on the computer monitor (Figure 38.2, far left). The day that we brought our tiny tarantula 3D map balsa and perspex stack models to the model room to show them to Hugh and Aaron is still alive in my memory. I came back to Venezuela on 14 December 1982, returning to Cambridge a few weeks later to complete the 3D reconstruction, to work with Roger and Jake Kendrick-Jones on activated tarantula thick filaments and to attend the Alpbach Workshop on Muscle to present the tarantula 3D reconstruction (Figure 38.1, right; Figure 38.2, left two images). I finally returned to Venezuela at the end of March 1983. We completed the work and wrote the paper which was published later (Journal of Molecular Biology, 1985. 184, 429–39). By then it was clear to me how fortunate I was to have the opportunity to spend these years in Cambridge and the LMB working in the intellectually free and exciting environment of Hugh’s lab, as well as collaborating with Roger on solving a scientific problem.

Figure 38.1. Raúl Padrón working at LMB in 1981 (left, photo by Roger Craig), and with Roger Craig (right, photo by Anne Anke Hennemann) attending the 1983 Alpbach Workshop on Muscle where they presented the first 3D reconstruction of negatively stained tarantula thick filaments (Figure 38.2, left and centre).

Figure 38.2. 3D reconstruction of negatively stained thick filaments of tarantula muscle plotted using a computer program written by Ed Egelman (far left: reproduced with permission from the Journal of Molecular Biology), with the putative arrangement of myosin heads superimposed (second left image). 3D reconstruction of frozen-hydrated tarantula thick filaments with the atomic models of four myosin interacting-heads motifs fitted along one helix (centre, photo by Lorenzo Alamo). The motif model appeared in a 2007 Venezuelan postage stamp (far right).

I frequently wrote notes on the key features of the LMB that I was discovering, hoping to implement them when I started my own lab in Venezuela. Not surprisingly, surrounded and fascinated by the high-quality science going on at the LMB, thoughts of returning to Venezuela faded every day. It was Carlo Caputo’s visit to London, and a day spent together at the British Museum, that brought me back to reality. I returned to his lab at the Biophysics and Biochemistry Centre at IVIC. IVIC regulations did not allow me to start a new independent lab. Despite that, Carlo was kind enough to allow me to work independently, as well as actively supporting my research. Nelly Panté, Hernando Sosa and Lorenzo Alamo were some of the bright young students who joined my lab then. We were all very busy setting up low-angle X-ray diffraction and electron microscopy facilities, which enabled us to keep studying the structure and function of tarantula thick filaments. My collaboration with Roger during these initial years in Venezuela was very fruitful, with the opportunity for my students, collaborators and myself to visit his lab at the University of Massachusetts Medical School in Worcester very frequently. In 1988 Lorenzo visited the LMB to work with Tony Crowther on the 3D reconstruction of activated tarantula thick filaments; Nelly and Hernando went to Brandeis to work with Carolyn Cohen and Hugh. Later on, Jose Reinaldo Guerrero, Carlos Hidalgo, María Elena Zoghbi and Guidenn Sulbarán went to UMASS, Julio Ortiz to the EMBL at Heidelberg and Antonio Biassuto to Oxford, while Maristela Granados and Reicy Brito stayed at the lab. I was able to run my lab with the support of several grants from MDA, as well as some timely support from NSF, NIH and TWAS. Surprisingly in 1997 a miracle happened, when I was designated as an International Research Scholar of the Howard Hughes Medical Institute (HHMI). Their support – that lasted until 2011 – allowed me to start a new centre at IVIC that year: the Center of Structural Biology. The first HHMI grant triggered local support, and I was able to buy a Philips CM-120 cryo-electron microscope. I certainly brought to the new centre all the lessons I learned from the LMB; including, last but not least, the importance of keeping one’s lab small.

During several years of work in close collaboration with Roger’s lab, we finally found a way to visualise intact – frozen-hydrated – relaxed tarantula thick filaments in the cryo-electron microscope. To our dismay, achieving very nice low-electron dose electron micrographs of tarantula thick filaments was not enough to calculate their 3D reconstruction, as the Bessel separation program that Tony developed in 1982 and other approaches we tried did not work well with these very low contrast images. It was a completely different image processing approach, devised by Ed Egelman in 2000, that finally enabled the calculation of the frozen-hydrated 3D-reconstruction in mid-2004. Roger noticed that the arrangement of heads was very similar to the structure observed in 1999 in 2D crystals of myosin molecules by the lab of Ken Taylor, another MRC alumnus. In 2005 we published the atomic model of the tarantula thick filament in the native (unfixed, unstained, hydrated) state (Nature 2005. 436, 1195–99); four of the six coauthors (Roger, Ed, Lorenzo and myself) were MRC alumni. The 3D map revealed that in the relaxed state the two heads of each myosin molecule interact with each other (Figure 38.2, centre), surprisingly with an arrangement similar to the structure observed by Taylor’s lab on the smooth muscle of a vertebrate. Our studies thus showed that this unusual, asymmetric head organisation, that we named the myosin interacting-heads motif (Figure 38.2, centre), was not an artifact of myosin isolation or crystallisation, but occurred in vivo. The conservation and uniqueness of this motif across the vast evolutionary distance between vertebrate smooth muscle and invertebrate striated muscle suggested that it was of fundamental functional importance for preserving the relaxed (‘off’) state in most or all muscles. Analysis of the interaction between the two heads has shown that one head (‘free’) physically blocks the actin-binding site of the other head (‘blocked’); this interaction simultaneously blocks the ATPase site of the free head. This head-head interaction immediately suggested a simple mechanism to explain relaxation – by switching off the two heads in different ways. Subsequent studies have demonstrated the presence of this motif in non-muscle cells, and in smooth and striated muscles of other species, confirming its widespread occurrence and the importance of tarantula thick filaments as a model system for studying thick filament structure and function. In further analysis we discovered a new intermolecular interaction between adjacent motifs (Journal of Molecular Biology, 2008. 384, 780–97) that would further stabilise the off-state. Our analysis of the head organisation, together with motility assays, sequence analysis and mass spectrometry observations, suggested a molecular model for muscle activation in which heads are phosphorylated sequentially by protein kinase C and myosin light chain kinase in a way that can explain both force development and post-tetanic potentiation in striated muscle (Journal of Molecular Biology, 2011. 414, 44–61).

In retrospect I feel happy and satisfied that Roger and I were able to pursue a project we started as postdocs in Cambridge 32 years ago: revealing the atomic structure of Hugh’s thick filaments. I am also happy to have successfully set up the new Center of Structural Biology at IVIC, the first structural biology centre in Latin America, in which I implemented the key lessons I learned at the LMB.

CHAPTER 39

Worms and Me, a Memoir (1981–86)

Cynthia Kenyon

Department of Biochemistry and Biophysics, University of California, San Francisco, CA

Former MRC-LMB postdocs told me not to go. It was not the same, they said. Francis Crick was in the US. Sydney Brenner was the LMB director now, and didn’t spend time in the worm lab anymore. You couldn’t get a job in the US from Europe. I was also advised not to work on C. elegans. Mark Ptashne said I was going down the worm tube. Developmental biologists (my future colleagues) were sceptical of the worm. Its developmental cell lineages (patterns of cell divisions and differentiations) were oddly invariant, and, for the most part, insensitive to loss of neighbouring cells. This had suggested that cell interactions and positional information, which were crucial for the development of other animals, were not very important for C. elegans. Instead, yeast-like cell-intrinsic asymmetric segregation mechanisms would predominate. So, C. elegans would turn out to be an evolutionary freak; cute perhaps, but irrelevant.

Of course, I did go, in late 1981. And I loved it, right from the start. My student and postdoc colleagues were amazing, and many went on to make great discoveries. Marty Chalfie and Andy Fire, who shared my lab room, have Nobel Prizes. Other stars included Ed Hedgecock, Barbara Meyer, Judith Kimble, Jim Priess, Tony Hyman, Iva Greenwald, Richard Durbin, and of course the LMB worm-lab heads Jonathan Hodgkin (who knew everything), John Sulston (the amazing one, mapping out the embryonic cell lineage right next to me) and John White (my inspiring friend, who taught me about artificial intelligence, worm neurons and cell biology). Gary Struhl was working with Peter Lawrence (who spoke eloquently, and had strong, interesting opinions). Gary’s rambunctious brother, Kevin, and my friend Linda Breeden worked with Kim Nasmyth (with whom I loved to talk science). I greatly enjoyed talking to all these people (most of them; though some, like Peter and Jonathan, were kind of scary to me), and to many others as well. It was the most stimulating environment I have ever known. To be honest, I should say that I wasn’t in the ‘in crowd’, which hurt; I remember one postdoc (who definitely was) apologising for not inviting me to a dinner party because she could only seat twelve. But I made many close, true friends at the LMB, who made me very happy, and still do.

A few times, I saw my advisor, Sydney Brenner. Sydney had thrown my application letter in the trash, recovering it only after he read my advisor’s letter of recommendation. So I was a little nervous about meeting him. After I had been at the MRC for about a month, Sydney invited me to his office, which was in another building. He gave me a book about developmental biology and asked me to come back when I had finished reading it. This was very kind, but I didn’t know anything about development. I had worked on bacteria in graduate school and I was still getting used to the idea of the nucleus. So I was scared to go back and never did. Some time later, Sydney asked me to his office again. He had heard that I was trying to transform C. elegans, by injecting DNA. He told me that if I succeeded, I should use transformation as a tool to study myosin. And, if my transformation attempts didn’t work, I should switch to flies, which could already be transformed, and study myosin. I didn’t want to study myosin, so I stole away and lay low. Somehow, though, Sydney kept tabs on me, because he wrote what must have been a nice letter of recommendation for me. Once I got my job, at UCSF, Sydney and I talked more frequently, but I was pretty much an advisor-less postdoc.

On the other hand, you could say I had twenty advisors: the Johns and Jonathan, the postdocs and students in the worm lab, Peter Lawrence, Kim Nasmyth, Mark Bretscher; all of whom knew more than I did and had their ideas and opinions. I wanted to study pattern formation. It was such an exciting field to me, and the more I learned, the more I liked it. How did a developing cell know what to become? Did it use localised positional cues or lineal (intrinsic) cues to find out? Were morphogenetic gradients real? What was an ‘organiser’?

At the time, Bob Horvitz’s lab had made amazing discoveries by studying C. elegans mutants. I knew about them because my graduate lab at MIT, the lab of Graham Walker, was right next door to Bob’s lab, and his students Iva Greenwald and Paul Sternberg were my close friends. I even sat in on some of Bob’s group meetings. I met Bob the morning he arrived in his new lab and found me there, camping out on my fold-out bed. Bob became my role model and great inspiration, and he is the one who suggested that I go and study worms with Sydney at the MRC, where he himself had trained. With Marty Chalfie and John Sulston, Bob had identified mutants that turned out to reveal genes for developmental timing. (One of these, lin-4, later became the founding member of the field of microRNAs.) Bob had also identified genes for vulval development. Vulval development, studied intensively in Bob’s lab, is now one of developmental biology’s great stories, and for these studies, along with studies of apoptosis, Bob received the Nobel Prize. One vulval gene, Iva’s gene lin-12, seemed particularly fabulous to me coming into the field, because loss-of-function and gain-of-function mutations in this gene caused opposite cell fate transformations in many different cell types. This was a quintessential switch gene and it told me there was logic to development.

I was drawn to C. elegans because by tracing individual cell lineages in mutants, and comparing them to the cell lineages of normal worms, you could pinpoint gene function at the level of individual cells. This level of resolution was analogous to having crystals to study protein structure. Bob and his lab members had made their discoveries by following cell lineages in mutants in which body structures were abnormal, and looking for recurrent features; for example, cells behaving as they would in younger animals. I wanted to discover my own new patterning strategy by analysing mutants.

At the LMB, Ed Hedgecock had been looking for mutants using Nomarski optics to see individual cells, and he suggested I try that too. He told me that of all the people he knew, I was the slowest to learn what worm cells looked like under the microscope. I remember finally being able to see a little cluster of neurons called the ‘postdeirid’, on the side of the animal. I was so excited that I ran down the hall and got my friend Barbara Meyer to come see it. But when she came, I could no longer find it. I learned a lot about pattern recognition during my postdoc. Soon I started screening for postdeirid mutants under the microscope, and I could detect the cell cluster as soon as it came into focus. My brain made postdeirids out of leaves in the forest, tiles on the floor, stars in the sky.

I found some interesting mutants. One, which I called the ‘yolk maker’, was later found to be a key sex determination gene in the worm. But I didn’t find anything that I thought would be completely new, which was what I was hoping for. One day, my lab mate Marty Chalfie told me about mab-5, a mutant isolated by Jonathan Hodgkin. These poor mutants, especially males, had many defects (‘mab’ stands for ‘male abnormal’), but one disorder seemed intriguing to me: the daughters of a cell called QL, which should migrate to the tail, instead migrated to the head. This seemed cool. The cells could still migrate, but they went to the wrong place. Also, the mutant failed to make six of its nine sensory rays (male mating structures). So the mutant could make rays, just not these six rays. There seemed to be an informational glitch in this mutant that could affect both migrating and stationary cells. So maybe if I could learn more about its other disorders, I could find some underlying logical structure, a method to its madness, and make a big discovery about the genetic programming of development.

I went into a small dark room and came out several years later. I was doing cell lineage analysis of the mab-5 mutant. It was hard. We all knew about the dreaded rudimentary mutant, a fly mutant that had tiny wings because, it turned out, of a purine deficiency. This was a prime example of a boring mutant. Would mab-5 turn out to be boring? If so, that was the end of me. I would disappear. This prospect was terrifying. I loved science and the thrill of discovery, and I had become committed to the idea of a life in science. Partly this was intrinsic to my nature, but it was also because of my good fortune in graduate school.

As a graduate student, I had carried out the first-ever promoter trap experiment, using a bacteriophage carrying a lacZ gene with no promoter. The eminent MIT bacteriologist Boris Magasanic reported on this phage after a Gordon Conference: you could insert it into your favourite gene, and then assay beta galactosidase (which was easy) instead of your own gene’s function (which could be hard). Graham’s lab was interested in the SOS response, in which low levels of DNA damage produced all sorts of changes in bacteria, including a stronger DNA repair capacity. After hearing Boris, I had the idea of inserting this little bacteriophage randomly into bacterial genomes, and then, using replica plating, asking whether I might find colonies that turned blue in response to low doses of mitomycin C. It worked. This was the first time that anyone had looked for genes on the basis of their expression, and I discovered a whole genetic regulatory circuit that is still yielding interesting insights into biology. This discovery enabled me to speak at two Gordon Conferences as a graduate student, and even tour the west coast giving seminars. (This was arranged, very kindly, by Keith Yamamoto, who later hired me at UCSF.) But now here I was in a dark room with an extremely messed up, possibly boring mutant.

Month after month I observed the mab-5 mutant’s development, cell by cell, looking for a pattern. But none came. I remember seeing copies of the journal Cell, where we all yearned to publish (though, I noticed, not the really great scientists, like John Sulston or Sydney Brenner). I would shudder and turn away; Cell was for other scientists, not for me. Meanwhile, science marched on. One day, the Drosophila Antennapedia gene was cloned. Antennapedia mutants were fascinating: they, and other homeotic mutants like them, had body parts in the wrong places. DNA Southern blot analysis showed that mammals had genes like Antennapedia, but worms didn’t. This was hugely significant for two reasons: first, it raised the possibility that developmental mechanisms were conserved evolutionarily, a wonderful, deeply fundamental new concept that we now take for granted. Second, it suggested that by working on worms, I was going to miss the boat. Worms didn’t have these fundamental patterning genes, probably because their bodies would turn out to be specified by lineal, rather than positional, mechanisms. Peter Lawrence suggested that I abandon the worm and study something interesting: Antennapedia. But I didn’t want to jump onto someone else’s bandwagon: I wanted my own. I pushed on with mab-5.

I followed all the lineages, or most of them, in the mab-5 mutant as it developed from the hatchling to the adult. The worms were beautiful to watch. Their little mouths took in their food, which streamed into their intestines. Their cells divided with little chromosome ballets. Just the textures of their cells and bodies were lovely to see. Sometimes, to trace long lineages, I had to watch an animal for many hours. I got to know one of the Cambridge A1 taxi drivers, and he would bring me takeaway food from a restaurant for dinner so I didn’t have to leave the microscope.

In mab-5 mutants, I found that cells still differentiated, but into the wrong cell types. For example, a cell called M made extra muscles instead of macrophage-like cells called coelomocytes. The cells that should generate those six missing male sensory rays, posterior members of a lateral line of cells called V cells, instead behaved like their counterparts in the hermaphrodite and made simple skin cells. So in some ways, the transformation was sexual (male to hermaphrodite), but on the other hand, the coelomocytes and the migrating Q cells were affected the same way in both sexes. No pattern.

Finally, after years of tortured uncertainty about mab-5 and my future, the epiphany came. It happened when I followed the development of the P cells, which run along the ventral surface of the animal (see Figure 39.1). P cells make the vulva, which was normal in mab-5 mutants, and they don’t make any other structures that are easy to see. So I didn’t know whether there would be anything wrong with them. To find out, I had to follow cell lineages. In normal worms, each P cell starts dividing the same way, but then differences emerge. Some cells die, some differentiate, some divide again. When it’s over, male P lineages are different from hermaphrodite P lineages, and in both sexes, anterior P lineages are different from posterior P lineages. This complexity let me unlock the secret of mab-5. I found that, as with the V cells, the posterior P lineages were defective in mab-5 males. But this time they didn’t look like posterior P lineages in the hermaphrodite. Instead they looked like male P lineages near the head. The same thing happened in hermaphrodites: posterior cells behaved like their normal anterior counterparts.

Figure 39.1. The six V cells and twelve P cells in the newly hatched animal are shown in the top left, and the basic P cell division pattern is shown in the top right. The fates of V and P cell descendants are shown below. S, skin cells. R, sensory rays. X, cell death. N, neuron. C, type VC neuron. Division symbol, cell that divides. Sexual vs. positional transformations cannot be distinguished in the V cell lineages, but they can be distinguished in the P cell lineages. The transformations in mab-5 mutants are most consistently interpreted as positional transformations, in which posterior V cells develop like their anterior counterparts, and posterior P cells develop like P2.

That was it. The sun came out and I had a model. mab-5 was a gene for the posterior body region. In its absence, posterior cells behaved like cells near the head. mab-5 wasn’t a sex determination gene, it had just seemed like one because the posterior is where male mating structures develop, and these go away in mab-5 mutants. Upon casual inspection, a bland male C. elegans posterior body region does resemble the normal hermaphrodite posterior body region, but not when you look closely. In fact, all of the V and P cell transformations could be interpreted as posterior-to-anterior transformations, whereas only some (the V cell lineages) could also be interpreted as sexual transformations.

Even the migratory changes could be interpreted as posterior-to-anterior transformations: There are two Q cells, one on the left and one on the right. QL and its descendants migrate posteriorly; QR and its descendants migrate anteriorly. The Q cells themselves migrated normally in mab-5 mutants, but the daughters of both QL and QR migrated to the anterior. Thus Q cell descendants in the posterior body region (QL’s descendants) were migrating like cells located more anteriorly (QR’s descendants). M was unique, so there was no way to know whether its odd lineage was a positional transformation or not.

The model was great, I thought. And even better, it created a fascinating paradox: As I said, C. elegans was thought to develop largely via cell-intrinsic lineage-specific controls, yet the cells in the posterior body region were not related by lineage. Thus the simplest model to explain my findings was to propose that C. elegans used positional signals to pattern its body axis after all. I was thrilled. I asked whether mab-5 itself might encode such a signal by using genetics to figure out whether normal cells might develop abnormally if other cells were mutant for mab-5. They didn’t, so if my model was correct, mab-5 was probably on the receiving end of a signalling pathway. However, by combining mab-5 with an additional mutation, I was able to slide the ray boundary towards the head or the tail by changing gene dosage, a phenomenon that really smacked of positional information. Bursting with passion, I wrote my article, and published it in Cell.

Several years later, at UCSF, we figured out what mab-5 was, by cloning it. Of all things, it was the worm ortholog of Antennapedia. I didn’t need to switch to flies to study Antennapedia, as Peter had wanted. I was already studying it. This was an important finding for science, but when I first learned mab-5’s DNA sequence, I had a terrible sinking feeling. After all my hopes of finding something new, I had just extended a field pioneered (beautifully) by others, to the worm. Darn. Nevertheless, the finding was intriguing. In fact, suddenly I was invited to all the major developmental biology meetings to talk about the worm’s version of Antennapedia. We studied this gene, and others like it, for many years. They were fascinating in many ways, and we made many interesting discoveries, but eventually I went into an intriguing new field in which I could be more of a pioneer (my dream all along): ageing. I won’t talk about these studies here, because this is an MRC memoir, except to say that it was at the LMB that the seeds of my ageing studies were planted.

Normal worms take three days to reach adulthood, at which point they begin their life’s work, making more worms. In the next three days, worms have 300 progeny. If you put a single adult hermaphrodite on a petri dish, three days later you can’t find it any more because it disappears into a sea of progeny. But one mutant I worked with had very few progeny. One day, probably in 1983 or so, I took a forgotten worm plate out of the incubator, and was stunned, totally stunned, to see that there were old worms on it. I had never thought about whether worms might get old. But they did. There they were, old worms. I felt sorry for them. Then that haunting, fear-of-dying feeling swept over me. I would get old too. It was a profoundly moving moment for me. As I said, I felt close to the worms, emotionally, having watched so many grow up, so this sensation went right through me. Then I thought, ‘You know, you could study this …’

Doing research at the MRC was thrilling and tumultuous. But not only did I need to discover something, I needed to learn about the whole field of development. To do this, I started a two-member journal club with my friend Maria Leptin, a postdoc who had come to development from immunology. A journal club of this size maximised one’s opportunity to talk. We read everything together: the history and concepts of development, described in books like the one Sydney had given me, as well as classic papers. We scoured the current literature. We met two or three times a week, over dinners at my home, in pizza restaurants, in a lovely hotel with a huge fireplace where nicely dressed men brought us tea and scones. On sunny days, we sat outside in the beautiful Orchard tea garden, with more scones and tea. We ended up with one compartmentalised mind. Maria remembered all the names of the animals, which I would forget. I didn’t mind or even notice this until I tried to talk about development with someone else. I loved the field of development, with its animals, its mathematical gradient models, and its strong personalities. I remember my tears of joy when Janni Nüsslein gave a seminar showing that the Drosophila patterning protein bicoid formed a gradient. There were morphogenetic gradients. You could see them. There they were.

But aside from science, England itself was wonderful. I began to feel, living there, that my soul was English. I fell in love with the country. I liked walking along the footpaths near the streams. I liked to work at night, when there was less arguing and bellowing, and I remember hearing the clear melodic song of the nightingale as I rode my bike home at dawn. On Saturdays, I went to evensong at King’s College to hear the boys’ choir. I wore pastel, earth-coloured wool sweaters from Cambridge shops and rode my bicycle around the countryside. The soft sunsets, also pastel, were so beautiful. Luckily for me, Gary Struhl arranged a trip to the opera for us postdocs soon after I arrived in England. We saw Don Giovianni, and I was hooked. I took to going to Covent Garden, on my own on the train, about every other week. I stayed in a little place called the Sandringham Hotel, on Hampstead Heath, and took walks on the Heath the next morning, before going back to Cambridge. It was all very romantic and satisfying.

Another source of happiness was my friendship with Barbara Meyer. I would often drink coffee with kahlua with Barbara at the Green Man (she drank Scotch whisky) and talk about science and scientists. We were such good friends that Kim Nasmyth asked me what I would do when she left. But the wonderful thing about the LMB was that there were more potential friends for me than I had time. I was part of a great swarm of LMB postdocs, many of whom were expatriate Americans. We often ate together, at Indian restaurants or often at my house. I lived with my other especially good friend, Tabitha Doniach, Jonathan’s graduate student, in a house in Newnham that we rented from John Gurdon. We served everyone large pots of macaroni and cheese with tomatoes, or sometimes something more elaborate like coq au vin. We invited other friends as well, often Tim Hunt from Cambridge. And the conversations! I had never really experienced the art of conversation before being in England. You needed to know something about a subject (say, Watergate), but more importantly, you needed to choose a side, any side, and defend it brilliantly. It was great fun.

Figure 39.2. Cynthia Kenyon in her back yard in England, c.1984.

In the end, I wanted to stay at the MRC and live in England. When a position opened up, I asked to stay, but I was turned down, because, they told me, Gary Struhl (who truly was exceptional) might accept it. So I went back to America with my wonderful English memories, and, at least so far, that has turned out well.

CHAPTER 40

Adventures at the Laboratory of Molecular Biology in 1984

Thomas D. Pollard

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT

A Guggenheim Fellowship allowed me to take leave from my position as professor and director of the Department of Cell Biology and Anatomy at Johns Hopkins Medical School to work for a year at the Medical Research Council Laboratory of Molecular Biology in 1984. This memoir will focus on the scientific work during that year. However, as for many other visitors to Cambridge, this adventure involved our whole family. We still debate who had the most fun and benefited the most from our time in Cambridge. Therefore, I asked the family to join me in reflecting how the experience influenced everyone’s lives.

SCIENCE

I was attracted to Cambridge by the opportunity to work with Hugh Huxley, one of my scientific heroes, whose work on muscle emphasised the importance of structure in understanding biological processes. Hugh kindly arranged for an Overseas Fellowship for me at Churchill College, which provided a beautiful flat for the family with a southerly view over the sports fields. Frances Huxley made sure that we felt welcome with a fridge stocked with food when we arrived early in January.

The main goal for the year was a high-risk attempt to visualise directly the motion of myosin cross-bridges on actin filaments by electron microscopy. Before embarking on this project, I wanted to make sure that something valuable would be accomplished during my visit, so teamed up with Alan Weeds to answer a central question concerning actin.

Since 1950 it had been known that actin polymerises into filaments and hydrolyses the ATP bound to each subunit. For years hydrolysis was thought to be coincident with incorporation of each actin subunit into a filament, but some experiments suggested that hydrolysis occurs some time after polymerisation. However, the timing was not clear. We designed a simple experiment to determine the timing. Since the reactions take place on a timescale of seconds, we worked out a routine to rapidly mix the samples, stop the reactions and measure the radioactivity associated with the phosphate cleaved from the ATP. At the end of each four-minute experiment we were breathless and relieved that no radioactivity had spilled. We discovered that actin hydrolyses ATP with a half time of about ten seconds, so when actin filaments are growing quickly, the ends have a zone of subunits with ATP. Our short paper was accepted promptly after submission in March 1984 (Pollard and Weeds, 1984). Subsequent work refined the measurements of the hydrolysis rate constant (Blanchoin and Pollard, 2002), established that the phosphate dissociated slowly from the filaments (Carlier and Pantaloni, 1986) and worked out details about how ATP hydrolysis and phosphate dissociation influence subunit association and dissociation at the two ends of the filaments (Fujiwara et al, 2009).

The question motivating the main project was: how does myosin produce force and motion? Hugh had established that myosin forms cross-bridges between the thick and thin filaments of striated muscle. Biochemists had mapped the biochemical pathway through the cycle of reactions of myosin with ATP and actin filaments and showed that myosin binds strongly to actin filaments in the absence of ATP. Hugh showed that in this strongly bound state the catalytic domain of myosin attaches to the actin filament at an angle of about 45°. ATP binding dissociates myosin from actin filaments and the myosin-ATP and myosin-ADP-Pi intermediates in the ATPase cycle exchange rapidly on and off actin filaments, as Hugh had documented by X-ray fibre diffraction. When the phosphate dissociates from the actin-myosin-ADP-Pi intermediate, myosin undergoes a force producing transition into the strongly bound actin-myosin-ADP state. One possibility was that this transition involves tilting of the myosin head from an angle around 90° to the 45° state. Alternatively, the myosin head could remain stationary while some other part of the cross-bridge produces the motion. We aimed to distinguish these two hypotheses.

The idea was straightforward: capture the ephemeral weakly-bound intermediates in the actomyosin ATPase cycle and view them by electron microscopy for comparison with the strongly bound 45° state. Our plan was to build a machine to mix and freeze protein samples on a millisecond time scale, allowing one to capture myosin-ATP bound to actin filaments and then prepare the specimens for electron microscopy by freeze fracturing and rotary shadowing. I did not have access to the technical expertise to build such a machine at Johns Hopkins, but Hugh was eager to support the development of the equipment and the famous MRC machine shop was up to the task. Over a period of several months machinist Chris Raeburn added a stopped flow mixer to a device that used jets of liquid propane to rapidly freeze the specimen (Figure 40.1, left). We experimented with many designs for the sample holder, which had to have very thin copper walls but also withstand the injection of the pressurised liquid sample. This technical problem was solved only after I returned to Johns Hopkins when a colleague referred me to a company that made us cylindrical copper flow cells with walls only 10 μm thick. The machine allowed us to mix ATP, myosin heads (subfragment-1) and actin filaments with a dead time of only 5 ms and to freeze the sample after a few milliseconds when the actin filaments were populated with myosin-ATP and myosin-ADP-Pi (Pollard et al, 1990). Completion of the experiments took several more years at Johns Hopkins.

Electron micrographs of replicas of the frozen samples (Figure 40.1, right) showed that the catalytic domain of myosin attaches to actin filaments at the same angle regardless of the bound nucleotide (Pollard et al, 1993). We concluded: ‘These observations suggest that the molecular motion produced by myosin and actin takes place within the myosin head at a point some distance from the actin binding site.’ Dave Thomas wrote a nice perspective on the work (Thomas, 1993).

Figure 40.1. Left, photograph of the stopped flow rapid freezing machine built by Chris Raeburn. (From Pollard et al, 1990). Right, electron micrographs of a freeze fracture replica showing ATP- myosin heads weakly bound to actin filaments. (From Pollard et al, 1993)

Details of the mechanism emerged five months after the publication of our paper, when the crystal structure of the myosin head revealed that the myosin light chains stabilise a rod-like connection between the myosin catalytic domain and the tail of the molecule, which forms the thick filament (Rayment et al, 1993a,b). Multiple crystal structures and elegant experiments with spectroscopic probes have established the light chain domain rotates about 90° between the actin-myosin-ADP-Pi and the actin-myosin-ADP state. We did not see this rotation, since the light chain domain was not preserved in our replicas, but our main finding that the catalytic domain is relatively stationary was correct.

Figure 40.2. Chris Raeburn in the lab with a stopped flow spectrophotometer that he built.

I am deeply indebted to the stimulating scientific community at the MRC lab for strengthening my resolve to use biophysical approaches to study cellular motility and cytokinesis over the past 25 years. I have also valued the many friendships that developed over lunch in the canteen at the LMB. The late Paul Sigler (visiting Cambridge from the University of Chicago) was a frequent companion at lunch. We were often amused when our English colleagues were surprised to learn that we were both MDs who had turned into biophysicists. I also recall with pleasure a fire drill one Saturday morning when the dozen individuals evacuating the LMB consisted of about equal numbers of British Nobel laureates and American professors on leave in Cambridge.

FAMILY

It was a joy for everyone to be embedded in British culture for the year. Patty organised the family activities including opera performances at Covent Garden and English National Opera, and visits to numerous London museums. Like everyone else in the country, we were glued to BBC TV for Torvill and Dean’s ice-dancing gold medal in the Winter Olympics. The fellowship at Churchill College introduced us to families from around the world and academic friends from Cambridge, although we never developed a taste for haggis in spite of Master Sir Will’s enthusiasm. Figure 40.3 shows some of our friends at our Fourth of July Independence Day party at Churchill, perhaps the first celebration of the event at the College. Our lives were enriched and perspectives broadened by day trips around Cambridge and longer trips to Wales, a winter trip to ski in Switzerland and summer trips to Germany, Italy and France.

Figure 40.3. Guests at the Pollards’ Fourth of July Independence Day party at Churchill College. Tom Creighton, Paul Sigler, Murray Stewart, Hugh Huxley and Alan Weeds.

Katie, aged eleven at the time, attended the Perse School for Girls. Despite various linguistic snafus and her complete lack of knowledge about netball and rounders, Katie quickly made friends with several classmates who remained pen pals for years to come. Her most vivid memories of the year are of break dancing, acting, sculpting punk hairdos and playing video games with children of the other international families in Churchill’s Sheppard Flats. She also remembers relishing new hobbies, such as horse riding, brass rubbing and English tea.

Dan, aged eight at the time, attended St Faith’s School in Cambridge. While St Faith’s presented some challenges, such as wearing a bright red coat and tie, eating potato chips with a fork and passing handwriting tests, Dan enjoyed the school’s sophisticated classical music listening class, as well as new sports like cricket, and track and field. Perhaps the biggest impact on Dan’s education that year was his newfound interest in history gained by hands-on experiences at Druidic ruins and medieval castles, sites older than his home country!

Acknowledgements

I thank the members of my family for contributing their recollections of our year in Cambridge. Katie is now faculty at the J. David Gladstone Institutes, University of California, San Francisco and Dan is a postdoc at the Division of Biological Sciences, University of California San Diego. Their year in British schools helped to propel them towards careers in science.

REFERENCES

Blanchoin, L., T.D. Pollard, 2002. Hydrolysis of bound ATP by polymerized actin depends on the bound divalent cation but not profilin. Biochemistry 41, 597–602.

Carlier, M.F., D. Pantaloni, 1986. Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments. Biochemistry 25, 7789–92.

Fujiwara, I., D. Vavylonis, T.D. Pollard, 2007. Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy. Proceedings of the National Academy of Sciences of the United States of America 104, 8827–32.

Pollard, T.D., A.G. Weeds, 1984. The rate constant for ATP hydrolysis by polymerized actin. FEBS Letters 170, 94–98.

Pollard, T.D., P. Maupin, J. Sinard, H.E. Huxley, 1990. A stopped-flow/rapid-freezing machine with millisecond time resolution to prepare intermediates in biochemical reactions for electron microscopy. Journal of Electron Microscopic Technique 16, 160–66.

Pollard, T.D., D. Bhandari, P. Maupin, A.G. Weeds, H.G. Zot, 1993. Direct visualization by electron microscopy of the weakly-bound intermediates in the actomyosin ATPase cycle. Biophysical Journal 64, 454–71.

Rayment, I., W.R. Rypniewski, K. Schmidt-Bose, R. Smith, D.R. Tomchick, M.M. Benning, D.A. Winkelmann, G. Wesenberg. H.M. Holden, 1993. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261, 50–58.

Rayment, I., H.M. Holden, M. Whittaker, C.B. Yohn, M. Lorenz, K.C. Holmes, R.A. Milligan, 1993. Structure of the actin-myosin complex and its implications for muscle contraction. Science 261, 58–65.

Thomas, D.D., 1993. Pollard to actomyosin: ‘Freeze! Don’t even move your head’. Biophysical Journal 64, 297–8.

CHAPTER 41

My Sabbatical at LMB (1985)

David Eisenberg

Institute of Genomics and Proteomics, University of California, Los Angeles, CA

In 1985 it was my good fortune to arrange a seven-month sabbatical at the LMB, with the then-director Aaron Klug. I arrived with the idea of collaborating with Andrew McLachlan. Both Andrew and Aaron asked me: ‘Why with Andrew?’ The answer was that I came with vague ideas about moving the concept of hydrophobicity from a residue level to an atomic level to create a simple way of estimating energies of solvation of proteins. Andrew had been a visitor to Caltech in the late 60s when I had been a postdoc there, and I recalled that he possessed a physicist’s knack for mathematical modelling. In Cambridge I knew what I wanted to do, and what physical measurements I wanted to harness into a theory, but I suspected I would become bogged down in setting up the proper equations. Luckily Andrew was willing to consult with me, provided it was over coffee or tea in the LMB canteen on the top floor. When I would sit next to him with my tea and set out my current puzzle, he would wrinkle his brow and say, ‘David, I think you should try such and such.’ Each of his suggestions would carry me forward for several days, until I would find myself stuck again, and I would have to seek out Andrew at tea time. Along the way I also received lots of helpful suggestions from Tim Richmond, whose room I shared, and from Arthur Lesk and Cyrus Chothia – all of whom had thought deeply about protein energetics.

The work came together after a few months, and for days I sat in the large computer-terminal room drafting a paper. One day Max Perutz asked, ‘David, what are you writing?’ I explained and he said he would like to read it. I passed him my draft and he returned it the next day with an encouraging note and numerous suggestions in neat red pen on how to use the English language. Andrew and I sent the final version to Nature, where according to Google Scholar it has received over 1,500 citations.

In one of our tea-time conversations, Andrew and Arthur Lesk got me interested in trying to write a computer program that could recognise all sequences of a given protein family, such as all immunoglobulins or all globins, without recognising any other sequences. After my initial failures, Andrew came up with the idea of a position-dependent score. That is, for example, a histidine residue would receive a high score for being placed in a sequence position where a histidine is often found in that protein family, and a lower score in other positions. This scheme, which we called a ‘profile’, worked better than my earlier attempts. When I returned to UCLA at the end of my sabbatical, Michael Gribskov, who had just joined our group from Wisconsin, improved my primitive program, and we were able to achieve fairly good success in recognition of distantly related sequences belonging to a given family. Griskov, Andrew and I published these ideas in PNAS, where they have also been cited over 1,000 times. Later, researchers at NCBI generalised the profile idea into the powerful program PSI-BLAST.

Later at UCLA, as a way of recognising if a very distant sequence is compatible with a given protein fold, Jim Bowie and I extended the concept of profiles into three dimensions. We used a position-dependent scoring table for assessing if a protein sequence is compatible with a given protein 3D structure. Soon after, Janet Thornton invented the graphic term ‘threading’ for such procedures. The next year Roland Luethy, Jim Bowie and I turned the procedure around, showing that it could detect errors in protein structures. These studies were in a sense extensions of the work that I had been involved with at the LMB.

My half-year in Cambridge was one of the most stimulating and enjoyable of my career. The whole atmosphere of the LMB was geared for discovery: an absence of red tape, an abundance of resources, a crowd of intelligent and curious colleagues, and a designed social system featuring a high concentration of brain power in a small canteen at coffee, lunch and tea every work day. Though I seemed to get more work done per unit time at the LMB than at home, I also seemed to have more time for social interactions. I enormously appreciated the collegiality and friendship that my family and I received from so many scientists there, including the McLachlan family, the Perutz family, the Klugs, my lab mates – Tim Richmond, Phil Evans and Clarence Schutt – and Arthur Lesk, Cyrus Chothia and Uli Arndt, as well as others.

List of Illustrations

0.1 Hugh Huxley, first skiing holiday, New Year 1949, near Kuhtai, Austria (on Max’s recommendation)

1.1 For a given X-ray reflection, addition of waves from different atoms in one molecule

1.2 For one X-ray reflection, addition of scattering vectors from a parent molecule Fo and an added heavy atom Fx to yield the observed experimental scattering from the heavy-atom derivative Fo+x.9

1.3 Phase-circle diagram for one X-ray reflection, using experimental amplitudes from the parent molecule O and the two heavy-atom derivatives X and M

1.4 Phase circles for three X-ray reflections from myoglobin at 6Å resolution, using parent myoglobin and five different heavy-atom derivatives

1.5 Lack-of-closure errors in phase analysis

1.6 Phase-angle probabilities plotted around a 360° phase circle of unit radius

1.7 Examples of phase determination for three X-ray reflections in the 2Å phase analysis

1.8 Section through the electron density of the haem group in the 2Å structure analysis, with an ideal framework superimposed

1.9 View through stacked Plexiglas sheets showing the haem group seen on edge and a view down the hollow F-helix that is attached to the haem by a His side chain

1.10 Density in an unrolled α-helix at 2Å

1.11 An accurate but simplified diagram of the folding of α-helices in sperm whale myoglobin from the 2Å analysis

1.12 The 2Å electron density displayed as coloured clips in a forest of steel rods mounted on a plywood base

1.13 Dr Frankendrew’s monster

1.14 Max Perutz as Noah, bringing his proteins safely through the deluge

2.1 The Medical Research Council Hut at the New Museum Site off Free School Lane

2.2 Composite view of the (FHgAc2 − FPCMB)2 correlation function between the HgAc2 and para-chloromercurybenzoate (PCMB) heavy-atom derivatives

2.3 The 5.5Å-resolution haemoglobin map. Section y = 1 showing long rods of density representing α-helices

2.4 The 5.5Å-resolution haemoglobin map. Section y = 9 showing long rods of density representing α-helices

2.5 Interpretation of the 5.5Å-resolution haemoglobin map

2.6 Max Perutz’s model of the 5.5Å-resolution map of haemoglobin

2.7 Dick Dickerson and Bror Strandberg with the myoglobin sorting board

2.8 John Kendrew and Max Perutz standing behind the forest of rods into which the first sperm whale myoglobin model was built in the basement of the Cavendish Laboratory

3.1 Robert G. Shulman

4.1 A sketch in projection of the heme region in (a) native metmyoglobin and (c) the azide derivative. The position of the difference peaks is depicted in (b)

5.1 Bill Dove

5.2 Alexandra Shedlovsky

6.1 Electron density map showing, in projection, the difference between DFP-inhibited and native α-chymotrypsin

7.1 Mike Reedy, 1965

7.2 Mike and Deirdre Reedy, dressed for St John’s May Ball, c.1965

8.1 Ignacio Tinoco in 1964 and 2010

9.1 Jon Beckwith, Roger Freedman and Tony Stretton, on the canteen balcony at LMB

10.1 Gerald Bricogne and Stephen Harrison en route to the CECAM Workshop on Virus Crystallography

11.1 Collection of geometric models exploring possible virus structures etc

12.1 Frank and Henriette (Jette) Foss

12.2 Frank and Matt Meselson

13.1 Programme from the 1641st concert of the Cambridge University Musical Club

14.1 Nonsense suppressors

14.2 Increase of tyrosine tRNA and suppressor activity in E. coli CA274 su- lac- amber following infection by φ80dSu+III.

14.3 Fractionation of φ80dSu+I tRNA by chromatography on DEAE Sephadex

14.4 T1 Ribonuclease fingerprint of the Su+ tRNA

14.5 Tyrosine tRNA E.Coli

14.6 Nucleotide sequence of the su+III tyrosine suppressor tRNA

14.7 John Abelson in the ‘ballet of protein synthesis’ as the 30s ribosomal subunit and Paul Sigler as magic magnesium in the LMB Christmas pantomime, 1967

15.1 David DeRosier at a meeting in honour of Aaron Klug

15.2 Joyce Baldwin, David DeRosier and Tony Crowther at a meeting in honour of Aaron Klug

18.1 Joan Steitz, Tom Steitz, Jerry Adams and Suzanne Cory in Switzerland, c.1968

19.1 Peter Moore

20.1 Mark Bretscher, Christine Roberts, Jeff Roberts and Joan Steitz

20.2 Tom Steitz and Mark Bretscher

21.1 Party at the Abelson cottage. Mark Bretscher, Jean Thomas, Margaret Clarke, Joan Steitz, Lynne Abelson, John Abelson and Brian Clarke

22.1 1983 Symposium of MRC Fellows

23.1 Vivianne Nachmias

24.1 Michael Levitt, c.1968

26.1 James Spudich and Hugh Huxley at Brandeis in 1996

26.2 James Spudich and his wife Anna at the May Ball in 1970

26.3 Interaction of tropomyosin-troponin with actin filaments

26.4 The first quantitative in vitro motility assay with purified actin and myosin

26.5 Subfragment 1 (S1) of myosin, the motor domain

26.6 In vitro motility taken to the single molecule level using the physics of laser trapping

27.1 Leonard S. Lerman

27.2 John D. Smith

27.3 Sidney Altman’s cottage in Barley

27.4 A description of tyrosine tRNA with locations of mutations of the amber and ochre anticodons at position 35

27.5 Analysis of mature and precursor tRNAs in a polyacrylamide gel

27.6 The secondary structure of a precursor to tyrosine tRNA

27.7 Several of the senior leaders in the MRC lab around 1967: Hugh Huxley, Max Perutz, Fred Sanger, Sydney Brenner, John Kendrew and Francis Crick

28.1 Takeyuki Wakabayashi

29.1 Henry Epstein

30.1 Mark Ptashne

31.1 Sarah Hitchcock-DeGregori, Joyce Baldwin and Betsy Goldsmith (formerly Heidner)

31.2 Sarah Hitchcock-DeGregori, Tom Pollard and Dennis Bray

33.1 Shneior Lifson, Arieh Warshel and Michael Levitt

33.2 Arieh Warshel’s wife Tami and daughters (Merav and Yael)

33.3 The QM/MM model of an enzyme active site

33.4 Snapshots from the simulated MD trajectory of the primary event in vision

34.1 J. Richard McIntosh

35.1 Paul Wagner, Werner Kühlbrandt and Jon Scholey

35.2 South Italian LMB lizard

35.3 Note left by Werner Kühlbrandt on Richard Henderson’s desk

35.4 Michael Rossmann, Werner Kühlbrandt, Nigel Unwin and Richard Henderson, in Stockholm, Sweden

36.1 Aaron Klug, Bob Diamond and Steve Lippard at a meeting in honour of Aaron Klug

37.1 Donald Engelman in front of a Lake Baikal poster

38.1 Raúl Padrón working at LMB in 1981, and with Roger Craig attending the 1983 Alpbach Workshop on Muscle

38.2 3D reconstruction of negatively stained thick filaments of tarantula muscle

39.1 The six V cells and twelve P cells in the newly hatched animal, and the basic P cell division pattern

39.2 Cynthia Kenyon in her back yard in England, c.1984

40.1 The stopped flow rapid freezing machine built by Chris Raeburn, and electron micrographs of a freeze fracture replica showing ATP- myosin heads weakly bound to actin filaments

40.2 Chris Raeburn in the lab with a stopped flow spectrophotometer that he built

40.3 Guests at the Pollards’ Fourth of July Independence Day party at Churchill College. Tom Creighton, Paul Sigler, Murray Stewart, Hugh Huxley and Alan Weeds

Index of Names

Abdel–Meguid, S.S. 40

Abdulaev, N. 312

Abelson, J. 119, 123–31, 150, 152–4, 161, 167, 174, 181, 187, 190, 192, 212, 232, 242

Abelson, L. 187

Aboul-ela, F. 83

Adams, J. 152, 154, 157–68, 181, 192

Adelman, M. 200, 222

Agard, D. 312

Alamo, L. 320, 321

Allison, W. 214

Altman, S. 58, 61, 130, 152, 229–42

Alver, E. 3, 4

Ames, B. 181

Amos, L. 174, 215, 244, 250, 263, 265, 266, 284, 285, 288, 300

Anderson, L. 49, 151, 192

Anfinsen, C. 252

Arditti, R. 92

Arndt, Uli 33, 94, 137, 193, 340

Aslund, F. 92

Astrin 165

Baldwin, Joyce 94, 96, 133, 218, 261, 265, 300

Baltimore, D. 146

Banaszak, L. 194

Barber, J. 302

Bardwell, J.C.A. 92

Bargmann, C. 239

Barnard, E. 186

Barnett, Leslie 25, 41, 48, 61, 111, 216

Barrell, B. 119, 150, 152, 159, 167, 179, 235, 258

Beckwith, J.R. 85, 91, 92

Beer, M. 123

Behar, K.L. 49

Belin, D. 92

Benaceraff, B. 318

Bennett, J.M. 5, 21, 72

Benzer, S. 91, 267

Berg, H. 289

Berg, P. 218, 237

Berger, J. 77, 95

Bernal, J. x, xi, 25, 27, 36, 38, 64

Bernhard, S. 69, 117

Berra, Y. 137

Biassuto, A. 321

Bicat, T. 118

Bijvoet, J.M. 33, 34, 39

Billeter, M. 154

Birktoft, J. 193, 212

Bishop, M. 165

Bixon, M. 272

Blackburn, E. 258, 265

Blake, C.C. 40

Blanchoin, L. 332, 336

Bleil, J. 195

Blow, David 9, 21, 26, 29, 34, 35, 36, 39, 40, 54, 55, 64, 65, 67, 68, 69, 70, 96, 116, 119, 145, 161, 177, 183, 185, 193, 212, 296, 302

Blumberg, W.E. 48, 49

Bodo, G. 21, 28

Bohr, H. 229

Bolle, A. 91

Bommel, V. 39

Bonneville, M. 287

Borisy, G. 152, 174, 192, 216

Bowie, J. 340

Bragg, Sir Lawrence 3, 13, 27, 28, 37, 38, 54, 63, 74, 120, 123, 140, 158, 161, 242

Branden, C. 52, 55

Branson, H.R. 39

Bray, D. 261, 263

Breeden, Linda 323

Brenner, Sydney 3, 25, 41, 42, 43, 44, 48, 54, 58, 59, 60, 61, 69, 85, 86, 87, 88, 89, 90, 91, 92, 94, 96, 109, 110, 111, 112, 113, 119, 121, 122, 124, 129, 130, 133, 140, 142, 144, 145, 152, 155, 158, 160, 161, 170, 178, 181, 184, 186, 192, 193, 199, 212, 229, 230, 233, 234, 236, 238, 239, 240, 241, 242, 252, 253, 255, 256, 267, 268, 269, 286, 287, 288, 308, 323, 324, 326, 329

Bretscher, Mark 41, 85, 142, 143, 151, 157, 158, 159, 160, 167, 177, 178, 179, 180, 181, 187, 190, 245, 286, 324

Bricogne, G. 96, 97, 98, 300

Brightwell, Jenny 316, 318

Brinster, R. 165

Brito, R. 321

Broad, A. 25, 29

Broda, P. 87, 91

Brown, Dan 181

Brown, G.S. 46, 49

Brown, Margaret 191

Brownlee, George 127, 145, 150, 152, 159, 161, 163, 167

Brownlee, Susan 160

Burdon, R.H. 237

Burgen, A. 212

Burkitt 165, 168

Burland, T.G. 61

Burnet, M. 164

Burridge, K. 263

Campbell 58

Campbell, Angela (Mott) 36

Capecchi, M. 141, 160, 162, 166

Caputo, C. 315, 320

Carlier, M.F. 322

Carlson 74

Cashmore, A. 190, 235

Casjens, S. 100, 101, 105

Caspar, D. 93, 95, 96, 100, 138

Cassoly, R. 48

Cate, J. 122

Cech, T. 229

Chalfie, M. 286, 323, 324, 325

Champe, S.P. 91

Changeux 45

Chen, K.S. 61

Chen, X. 61

Chiu, W. 300

Chothia, C. 254, 339, 340

Chu, S. 226

Clark, J. 227

Clark, Brian 119, 128, 131, 151, 158, 159, 166, 167, 172, 187, 190

Clark, Margaret 187

Clarke, Margaret 222, 228

Clipson, L. 61

Cochran, W. 27

Cohen, Carolyn 320

Cohen, G. 86

Collard, Jill 36, 66

Coller, E. 39

Coombs, Susan 191

Cooper, Joan 213

Corey, R.B. 16, 39, 40

Cory, Suzanne 152, 154, 157–68, 181

Cotton, F.A. 306

Coulson, A. 259

Cowling, M. 294

Cox, A. 261

Cox, Joyce 94, 193

Craig, R. 316, 318, 319, 320, 321, 322

Creighton, Tom 254, 281, 335

Crick, Francis Crick, Francis 3, 9, 21, 25, 29, 39, 41, 42, 43, 44, 48, 54, 58, 59, 60, 61, 69, 70, 80, 89, 94, 109, 111, 113, 116, 119, 133, 137, 140, 142, 144, 145, 151, 153, 155, 158, 160, 162, 170, 177, 184, 185, 192, 193, 204, 205, 206, 230, 236, 238, 240, 242, 245, 252, 253, 255, 259, 264, 268, 293, 299, 323

Crowther, Tony 96, 133, 137, 140, 174,193, 284, 300, 320

Cruikshank, D. 2

Cullis, Anne 25, 29, 39

Cusick, M. 195

Cuzin, F. 61

Czerwinski, E.W. 65, 70

Dahlberg, J. 119, 122, 130, 149–55, 161, 163, 174, 181, 190, 192

Darie, C. 195, 196

Darnell, J.E. 237, 242

Darwin, C. 34, 201

Davidson, B. 117

Davies, D. 13, 14, 21, 67, 68, 80, 252

Davis, S. 264

Dawes, Jill (neé Collard) 36, 66

Delbruck, M. 101, 112, 113, 267

DeLozanne, A. 222

DePamphilis, M. 195

Derman, A. 92

DeRosier, David 133–40,152, 153, 172, 174, 192, 217, 220, 263, 284

Diamond, Bob 95, 98, 140, 193, 206, 300

Dickerson, R. 1, 20, 21, 23, 24, 30, 34, 36, 39

Dintzis, H. 21, 38

Dixon, J. 214

Dodson, E. 40

Dodson, G. 46, 49

Donelson, J. 258

Doniach, T. 330

Doty, P. 123

Dove, W. 57, 58, 61

Drake, J. 110

Drenth, J. 37

Dube, S. 150, 159, 167

Durbin, R. 323

Dworkin, M. 101

Ebashi, S. 219, 244

Eckstein, F. 307

Edelman, G. 286

Edgar, R. 110, 113, 267–9

Edmundson, A. 52

Edwards, R.J. 77

Egelhoff, T. 225

Egelman, E. 201, 318, 321, 322

Eigen, M. 208

Einstein, A. 229, 230, 243, 276, 302

Eisenberg, D. 339, 340

Eisenberger, P. 45, 46, 49

Engelman, D. 311–13

Ephrussi, B. 88

Epstein, Henry 251–6

Epstein, R. 91, 110, 111, 124, 267

Erickson, H. 192, 200, 217

Euteneuer, Ursula 286, 288, 289

Exupéry, A. de S. 217

Evans, Phil 249, 311, 340

Fankuchen, I. 64

Faruqi, Wasi 317

Fellner, P. 150

Fenn, R.H. 40

Fermi, Judd 46, 49

Fernandez-Moran, H. 316

Ferry, Georgina 31, 36, 38, 39

Fersht, Alan 212, 252, 254

Fiddes, J. 258

Finch, John 96, 97, 98, 100, 136, 137, 138, 139, 173, 193, 220, 222, 228, 244, 263, 284, 299, 300, 308

Finch, L. 158

Finer, J. 226

Fire, A. 323

Fisher, R.A. 111

Fishpool, Rita 256

Florey, H. 76

Florman, H. 195

Fogg, Jan 286

Fordham, Joy 264

Foster, N. 227

Frangioni, Blas 151

Frank, Joachim 300

Franklin, N.C. 61

Franklin, Rosalind 3, 100, 161, 266

Freedman, R. 58, 85, 89, 110, 124

Freifelder, D. 123

Fujiwara, I. 332, 336

Fuller, Michael 52, 96, 192, 238, 244, 263, 318

Fuller, Steve 301

Gabriel, A. 317

Gaeda, K. 315

Gait, Mike 308

Galkin, V.E. 200

Gamow, G. 230, 242

Garen, A. 91, 124

Garrett, R. 292

Garza, A.G. 103, 106, 107

Gefter, Malcolm 124, 131, 152, 190, 192, 193

Gellen, Marty 118

Gerhart, John 87

Gesteland, R. 122

Gething, M.J. 265

Gibbons, I. 284, 288

Gibson, Q. 45, 48

Giglio, I. 103

Gilbert, P. 204

Gilbert, W. 69, 162, 178

Gjedde, A. 49

Glauert, Audrey 75

Glaeser, R. 298, 300

Goaman, Gwynne 94

Gold, L. 287

Goldstein, L. 77, 287

Goodman, H. 119, 122, 124, 152, 153, 154, 161, 163, 192

Gorini, L. 90

Gould, M.N. 61

Green, D. 21, 38

Greenwald, I. 323, 324

Greer, J. 193

Greve, J. 195

Griffin, Beverly 150

Grimly, C. 92

Grissom, Paula 287, 289

Grodzicker, T. 92

Grodzins, L. 242

Gronewold, T. 103, 104, 106

Guenet, J.L. 61

Gull, K. 61

Gurdon, John 155, 318, 330

Gussin, G. 122, 141

Haag, J.D. 61

Hagen, D.C. 102

Hamilton, W. 37

Hammerschlag, R. 189

Hanson, Jean 244, 262, 317

Harding, M.M. 40

Harker, D. 8, 9, 21, 28, 29, 39

Harris, Ieuan 116, 117, 119, 121, 151, 170, 212

Harrison, S. 93, 97, 99

Hart, Dave 137

Hart, R. 3, 4, 9, 19, 21, 137, 140

Hartley, Brian 69, 117, 119, 151, 153, 185, 186, 212, 264

Hartman, C. 61

Haselgrove, John 174, 193, 245, 246, 263, 319

Hastings, W. 218

Hatano, S. 199, 201, 222, 228

Hawking, S. 180

Hawthorne, W. (Sir Will) 335

Hayder, F. 49

Hayes, W. 86, 87

Hayward, W.S. 165

Hayward, Len 137

Heath, E. 211

Hedgecock 323, 325

Heidemann, R. 285, 288

Henderson, Richard 55, 68, 70, 119, 174, 183, 187, 193, 212, 244, 263, 264, 278, 284, 296, 299, 300, 301, 302, 303, 304, 308, 311, 312

Hennessey, J. 227

Henson, J. 76

Herman, R. 268

Hershey, A. 124

Heuser, J. 242

Hidalgo, C. 320

Hightower, R.C. 200, 201

Hindley, J. 154

Hinshelwood, C.N. 267

Hirose, K. 249

Hirsh, D. 130, 152, 181, 190, 192, 287, 288

Hitchcock-DeGregori, Sarah 261–6

Hodgkin, Alan 76, 244

Hodgkin, Dorothy 23, 25, 38, 40, 266

Hodgkin, Jonathan 23, 25, 34, 36, 105, 138, 140, 239, 242, 323, 324, 325, 330

Hoeschele, J. 306

Hogle, J. 95

Hogness, D. 218

Hohn, T. 100, 105

Holley, R.W. 126, 128, 159, 167

Holmes, Ken 51, 55, 77, 78, 94, 95, 96, 137, 139, 193, 317, 337

Holmes, Mary 55

Holtzer, H. 202, 222, 228

Hopfield, J. 45, 48

Hopkins, J. 92

Hoppe, W. 140

Horvitz, R. 59, 61, 268, 286, 324

Housman, D. 147, 148

Houston, R. 294

Huang, Alice 146

Hunter, A. 61

Hurwitz, E. 258

Huxley, A.F. 76

Huxley, Frances 216, 331

Huxley, Hugh 54, 71, 72, 73, 74, 75, 76, 77, 94, 96, 99, 100, 119, 170, 171, 172, 173, 174, 193, 197, 198, 199, 200, 201, 202, 212, 215, 216, 217, 219, 220, 222, 227, 228, 240, 244, 245, 246, 250, 254, 255, 262, 263, 284, 300, 308, 309, 315, 316, 317, 318, 319, 320, 321, 322, 331, 332, 335, 336

Hyman, A. 323

Ingram, Vernon 21, 38, 134, 135, 252

Ippen, K. 92

Irving, A.A. 61

Ishikawa, H. 201, 202, 228

Jacob, F. 59, 61, 86, 87, 88, 89, 91, 92, 267

Jakes, R. 179, 263

Janin, J. 254

Jeffery, Barbara 40, 55, 64, 65, 70

Jeffrey, G. 2

Jeppeson, P. 163

Johnson, B. 307

Johnson, J. 307

Johnson, Louise N. 40, 55, 61, 94, 212

Jones, Alwyn 98

Jones, Graham 117

Josephs, R. 192, 216

Jovine, L. 195, 196

Judson, H.F. 58, 61

Jukes 179

Julien, B. 104

Kadokura, H. 92

Kaiser, A.D. 99–107, 218, 219, 237

Kaplan, Nathan O. 189, 214

Kaplan, S. 89, 92

Keilin, D. 52

Kendrew, John 1, 3, 4, 5, 9, 13, 14, 18, 19, 21, 24, 25, 26, 27, 28, 30, 33, 36, 38, 39, 52, 52, 53, 55, 59, 63, 79, 89, 94, 95, 98, 119, 121, 171, 185, 191, 192, 193, 204, 205, 206, 207, 208, 209, 251, 299

Kendziorski, C.M. 61

Kenyon, Cynthia 323–30

Khorana, G. 94, 151, 237, 309, 312

Kim, S.K. 103

Kimsey, H.H. 102, 103, 104

Kincaid, B. 45, 49

King, J. 102, 104, 212, 215

Klug, Aaron 3, 54, 77, 80, 89, 93, 94, 95, 98, 99, 100, 119, 133, 135, 136, 138, 139, 140, 151, 155, 161, 171, 172, 193, 205, 206, 207, 209, 212, 217, 220, 239, 241, 244, 245, 249, 275, 284, 294, 299, 300, 306, 307, 309, 311, 315, 318, 339, 340

Knappeis 74

Koenig, D.F. 40

Kornberg, A. 55, 100, 107, 218, 237

Kornberg, Roger 206, 245

Koshland, D. 44, 45, 185

Krimm, Marilyn 55

Krimm, Sam 55

Krogh, A. 165

Kroos, L. 103, 104, 106

Khrushchev, N. 76

Kuhlbrandt, W. 291, 304

Kuspa, A. 102, 106, 107

Kwong, L.N. 61

Laemmli, U. 212, 255

Lake, J. 300

Lamfrom, Hildegard 60

Landy, A. 119, 124, 131, 150, 152, 161, 167, 181, 190, 192

Lapiccirella, A. 279

LaRossa, R. 102, 106

Laube, B. 61

Lawrence, Peter 323, 324, 326, 328

Leberman, R. xii, 55, 93, 193

Leberman, Pat 55

Lehman, W. 218, 227

Leptin, M. 329

Lerman, L. 59, 230, 231, 242

Lesk, A. 339, 340

Leslie, Andrew G.W. 40, 248

Levinthal, C. 69

Levitt, M. 203–10, 254, 272, 281

Lewis, J. 307

Li, Shumin 256

Lifson, S. 204, 205, 207, 271, 272, 280

Lindberg, U. 263

Linck, R. 245

Lippard, S. 305–09

Lippman, F. 214

Lipscomb, W. 1, 3, 19, 20, 23, 119, 183

Lipson 2, 23, 95, 172

Lira, S. 195

Litscher, E. 195, 196

Liu, C. 195

Lodish, H. 119, 141–8, 151, 152, 161, 181, 192

Loewy, A.G. 198, 199, 202

Loftus, S. 295

Love, W. 123

Lowery, C. 92

Lowey, Susan 200

Lowy, J. 76, 244

Luft, J. 72

Lund, Elsebet 154, 190

Luria, S. 69

Lutter, L. 264, 306

Luzzati, V. 315, 317

Lye, R.J. 287, 289

Lymn, R. 228, 262

Magasanic, B. 326

Magnussen, S. 212

Mair, G.A. 40

Mallett, F. 137

Maniatis, T. 257–9

Manoil, C. 92, 102, 106, 107

Manstein, D. 225

Marcker, K. 119, 150, 158, 159, 162, 166, 167, 212

Markert, C. 238

Martin, S. 145, 152, 161, 190

Mason, G.F. 49

Masuda, T. 101, 106

Mateu, L. 315

Matsuura, Y. 249, 250

Matthews, B. 55, 63, 65, 67, 68, 69, 70, 116, 119, 214, 312

Mathews, F.S. 55

Mathews, M. 190, 212, 213

Matthias, A. 294, 295

Mayer, A. 49

McClain, W. 234, 235, 237

McGovern, K. 92

McIntosh, R. 283–9

McKellen, I. 217

McLachlan, A. 300, 312, 339, 340

Medlin, H. 63

Mellema, J. 245

Menninger, J. 110

Mertz, J. 265

Meselson, M. 109, 112, 113, 231, 232, 242

Meyer, B. 323, 325, 330

Mignot, T. 105, 107

Miller, J.H. 92

Milligan, R. 296, 301, 337

Milstein, C. 119, 151, 161, 166, 212, 239, 252

Moazed, D. 122

Moffat, J.K. 55, 174, 190, 193

Mommaerts, W. 75

Monot, J. 45, 46, 86, 91, 92

Monteverdi, G. 268

Moody, M. 140

Moore, P. 122, 137, 152, 154, 169–75, 192, 217, 220, 263

Moore, Stanford 117

Mooseker, M. 285, 289

Mott, Angela 36

Muhlethaler, K. 302

Muirhead, Hilary 9, 25, 29, 39, 183, 193

Murphy, C. 225

Murray, John 263

Murray, Ken 150, 161, 164

Murray, Noreen 112, 113, 164

Nachmias, Vivianne 197–202

Nagai, K. 244

Nakata, A. 112, 113

Nan, B. 105, 107

Nasmyth, K. 323, 324, 330

Needham, J. 180

Neel 165

Newton, Austin 90, 92

Newton, I. 116, 205

Newton, M.A. 61

Nicholls, J. 315

Nirenberg, M. 58, 94, 124, 158

Nixon, R. 261

Nobbs, C.L. 55

Noller, H. 115–22, 130, 151, 152, 154

Norman, E. 137

North, A.C.T. 33, 39, 40, 67, 70

Nossal, G. 164

Nüsslein, J. 329

O’Halloran, T. 307

Ogawa, S. 44, 45, 46, 48, 49

Oosawa, F. 199, 201, 228

Orgel, Alice 204

Orgel, L. 89, 204

Ortiz, I. 256

Ortiz, J. 320

Osborn, Mary 181

Ovchinnikov, Y. 311, 312

Padrón, R. 315–22

Pais, A. 230, 242

Palmiter, R. 165

Panté, Nelly 320

Pantaloni, D. 332, 336

Paoli, J. 49

Pardee, A. 85, 86, 87, 91

Parkinson, Jean 193

Parrish, R.G. 21

Parry, D. 246

Patel, A.B. 49

Pauling, L. 16, 24, 26, 37, 39, 40, 58

Pearse, Barbara 264, 265

Peerdeman, A.F. 39

Peisach, J. 48

Pepinsky, R. 2

Perona, R. 195

Perrin, D. 91

Perutz, Gisela 99, 150, 184, 193, 264

Perutz, Max 1, 3, 4, 8, 18, 21, 24, 25, 26, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 44, 45, 46, 48, 49, 55, 59, 63, 64, 69, 73, 89, 93, 94, 99, 115, 116, 119, 121, 135, 145, 160, 171, 175, 181, 183, 184, 187, 189, 191, 193, 203, 204, 205, 206, 207, 208, 209, 210, 212, 217, 229, 239, 240, 246, 248, 249, 252, 254, 259, 265, 276, 278, 279, 281, 291, 292, 293, 294, 299, 300, 302, 308, 317, 339, 340

Pfarr, C.M. 287, 289

Phillips, D. 3, 4, 13, 21, 30, 33, 36, 37, 40, 55, 67, 68, 70, 94, 120, 121, 274

Phillips, S. 51

Phizackerley, P. 137

Pickvance, S. 59

Pinkerton, Mary 25

Plamann, Lynda 102, 106, 107

Platt, J. 58, 61

Pollard, Dan 336

Pollard, Katie 336

Pollard, T.D. 201, 202, 222, 228, 261, 331–7

Pope, B. 263, 320

Porter, K. 283, 284, 285, 287, 289

Potts, Mary 154

Prescott, D. 287

Priess, J. 323

Pringle, J.W.S. 77

Prinz, W. 92

Ptashne, M. 257–9, 323

Puck, T. 230

Purcell, E. 51

Rabbitts, T. 164

Raeburn, C. 332, 333, 334

RajBhandary,T. 147, 148

Ramakrishnan, V. 186, 292

Rayment, I. 225, 226, 228, 334, 337

Reagan, R. 144

Redgrave, Vanessa 153

Reedy, Deirdre 73

Reedy, M. 71–8

Reedy, Mary 78

Reichenbach, H. 101, 107

Retzias, G. 284

Rhodes, Daniela 265, 308

Rice, Condoleezza 227

Rich, A. 29, 42

Richards, F. 37, 98, 186, 214

Richardson, J.P. 92

Richmond, T. 307, 339, 340

Rigby, P. 190, 212, 252, 256

Roberts, C. 177

Roberts, J. 177, 181

Robertson, D. 78

Robertson, H. 147, 148, 190, 235, 236, 238, 241

Robertson, M. 23, 25, 38, 241

Robinson, Hester 294

Roller, R. 195

Rossmann, M. 2, 5, 6, 7, 9, 23–40, 54, 64, 65, 70, 96, 122, 213, 214, 300, 304

Rothman, D.L. 49

Rothman, Jim 146, 148

Rowe, A. 76, 284, 288

Rowland, Eva 52

Rownd, R. 89

Ruppel, K. 225

Rushton, Barbara 306

Russell, R.L. (Dick) 124, 129, 131, 192, 242

Rutherford, E. 18, 25

Rutter, B. 219

Ryle, M. 27

Sanger, Fred 59, 69, 80, 89, 117, 119, 120, 127, 128, 130, 142, 145, 149, 150, 151, 154, 155, 158, 159, 160, 162, 163, 164, 166, 167, 171, 178, 179, 184, 192, 212, 232, 234, 238, 239, 240, 242, 249, 252, 253, 254, 257, 258, 264, 299, 308, 318

Sarabhai, A. 58, 89, 119

Saraste, M. 302

Sargent, P. 223

Sarma, V.R. 40

Sarwar, Z. 103, 107

Satir, P. 284, 289

Scaife, J. 87, 90, 91, 92

Schachman, H. 18

Schatz, P. 92

Schedl, P. 237

Schedl, T. 61

Schlesinger 237

Scholey, J. 289, 291, 301

Schmidt-Nielsen, Bodil 265

Shulman, R.G. 41–9

Schutt, C. 263, 300, 311, 340

Sedat, J. 258

Sharp, P. 307

Shedlovsky, Alexandra 57–61

Sheetz, M. 223, 224, 228

Shen, J. 49

Shih, W. 225

Shirakihara, Y. 249

Shooter, E. 223

Shore, V.C. 21

Schultz, R. 195

Sibson, W.R. 49

Siddiqi. O. 91

Sigler, P. 55, 66, 67, 68, 70, 94, 116, 119, 130, 131, 145, 161, 190, 191, 192, 214, 334, 335

Signer, E. 89, 92

Simmons, R.M. 226, 228, 317

Singer, Mitchell 102, 103

Sistrom, W. 86

Smillie, L. 69

Smith, John 89, 109, 119, 124, 130, 131, 145, 152, 152, 167, 190, 193, 231, 232, 234, 236, 238, 239, 242, 268

Soll, L. 239

Sosa, H. 320

Sperling, Ruth 244

Spector, P. 118

Spudich, J. 174, 192, 212, 213, 215–28, 244, 245, 263

Squire, J. 246

Staehelin, A. 287

Stahl, F. 60, 75, 109–13

Stark, B. 237

Stark, G. 218

Stein, W. 117

Steinberg, C.M. 91, 113

Steinrauf, L. 24

Steitz, Joan 119, 130, 142, 144, 145, 151, 152, 154, 157, 161, 174, 177–181, 187, 190, 192, 212, 213, 217

Steitz, Thomas 119, 130, 144, 152, 154, 157, 161, 174, 175, 177, 181, 183–7, 189, 190, 192, 193, 212, 213, 217, 292, 300

Sternberg, B. 324

Stevenson, K. 153

Stewart, E.J. 92

Stewart, Murray 244, 248, 249, 250, 263, 318, 335

Stombaugh, N.A. 49

Strandberg, B. 3, 4, 5, 13, 14, 21, 29, 24, 30, 34, 35, 39

Strasser, A. 165, 168

Streisinger, G. 267

Stretton, A. 54, 58, 85, 89, 92, 94, 239, 242

Stroud, R. 312

Struhl, G. 323, 329, 330

Stryer, L. 51–5, 218, 251, 252

Sulbaran, G. 320

Sulston, J. 59, 61, 239, 242, 268, 323, 324, 326

Sun, M. 105, 107

Sutoh, K. 247

Suzuki, M. 249

Svergun, D. 247

Schwartz, D.O. 92

Sweet, R. 217

Szent-Györgyi, A.G. 218, 219, 220, 227, 255, 262

Taddei, C. 296

Takanami 179

Tanner, T. 121

Taylor, C.A. 172

Taylor, E. 200, 201, 222, 226, 228, 262, 285

Taylor, K. 78, 298, 322

Taylor, Susan 211–14, 217

Thatcher, Margaret 294

Thomas, C.A. 123, 130

Thomas, D. 333, 337

Thomas, Jean 187

Thomson, J.J. 24

Thomson, N. 256, 286

Tilney, L.G. 285, 289

Tinoco, I. 79–84

Tissières, A. 121, 163, 170

Titus, Margaret 225

Tocchini-Valentini, G. 110, 119, 130

Todd, R. 308

Tomlin, S. 63

Torrealba, J. 61

Toyoshima, C. 228

Toyoshima, Chikashi 249, 250

Toyoshima, Yoko 225, 228

Travers, Andrew 152

Tregear, R. 77

Trikojus, V. 158

Traut, R. 122

Tsukihara, T. 40

Tullius, T. 308

Ullman, A. 92

Unwin, Nigel 174, 204, 244, 249, 263, 284, 285, 295, 296, 297, 298, 299, 300, 301, 302, 304, 311

Uyeda, T. 225

Vaisberg, E.A. 287, 289

Vale, R. 223, 228

Varani, G. 83

Varmus, H. 165

Vazquez, Monica 195

Vaux, D. 165, 168

Venter, C. 214

Vernon, C.A. 40

Vibert, Peter 319

Villa, P. 320

Villegas, J. 315

Wakabayashi, T. 243–50, 263

Wakabayashi, Katsuzo 247

Wakabayashi, Ken-ichi 249

Walker, G. 324

Wallace, Bonnie 312

Waller, J.L. 61

Warrick, H. 104, 105, 106

Warshel, A. 204, 205, 207, 271–82

Wassarman, P. 189–96

Waterston, R. 286

Watson, H. 19, 51, 52, 53, 54, 55, 68, 117, 189, 190, 191, 193, 194

Watson, Jim x, 3, 34, 58, 61, 86, 121, 122, 141, 142, 153, 160, 161, 162, 163, 170, 177, 178, 293, 299

Watts-Tobin, R. 48, 61, 242

Watt, Susan 220, 227

Weber, G. 254

Weber, K. 187

Webster, B. 141

Weeds, Alan 170, 200, 216, 220, 244, 254, 255, 263, 266, 331, 332, 335, 336

Weeds, Stella 216

Weichert, J. 61

Weigle, J. 100, 107, 112, 113

Weinzierl, J. 217

Weisenberg, R. 285, 289

Werhli, E. 302

Westergaard, O. 212

Wheatley, P. 1, 2, 3, 20

Wheeler, D. 27

White, J. 253, 268, 286, 323

Wickner, W. 101, 107

Wilcox, D. 161

Wilkerson, J. 233

Wilkes, M. 26

Wilkins, M. xii, 293, 311

Wilkinson, A.J. 49

Williams, Z. 195

Willingate, M. 295

Wilson, Benny 115

Wilson, Bright 272

Wittmann, H.-G. 291, 292

Woese, C. 122

Wollman, Elie 86

Wood, W. 242, 267, 268, 288

Woollard, A. 317

Wray, W. 319

Wuthrich, K. 44, 48

Wyckoff, H. 21,38, 245

Wyman, J. 45

Xu, S. 316

Yamamoto, K. 326

Yamane, T. 48

Yan 179

Yanagida, T. 224, 228

Yaniv, M. 161

Yanovsky, C. 61, 218, 219

Yates, J.L. 61

Yonath, A. 292

Young, Dora 189

Yusupov, Gulnara 122

Yusupov, Marat 122

Zachau, H. 126

Zadrazil, S. 124, 131

Ziff, E. 258

Zinder, N. 142

Zipser, D. 89, 90, 92

Zubay, G. 92, 198, 202

Index by Subject

Acanthamoeba castellanii 222

acridine-DNA complexes 230

acridine-induced mutants of tRNA 231–2, 235

actin xi, 43, 72, 76–7, 171–4, 197, 200–1, 216, 219–24, 225–6, 243–9, 256–63, 298, 317–18, 322, 331–4

2Å resolution structure 248

actin-myosin interaction 220, 246

actin-profilin 263, 336

actin-tropomyosin complex 245

azimuthal shift of tropomyosin 246

calcium-induced changes 244, 245

cytoplasmic actin 248

DNase I depolymerises actin 263

dynamic polymerization 243

filaments xi, 72, 200–01, 219–24, 243, 245–9, 298, 317, 331–3

fluorescent actin 224, 228

hydrolyses ATP 332

long pitch grooves of the actin 324

myosin-binding site on actin 246–7

paracrystals of actin 221, 245–6, 262

purification 220

second layer-line 246

second helical reconstruction of filaments 220

tropomyosin-binding site 248

afternoon tea 60, 119, 145, 150, 180, 216, 217, 218, 259

ageing studies 199, 328

allosteric transition 48, 252, 256, 279

Alpbach 318, 320, 321

amber

anticodon 234, 236

chain termination signal 89

nonsense mutation 54, 142

mutant 110, 111, 112, 113, 125

mutation 88, 129, 143, 144, 148, 267

stop codon 94

suppressor 87, 89, 124,126, 128, 131, 167, 231

amazing group 193

American football 192

Aminoff Prize 299

aminoacyl-tRNA synthetase 141, 142, 185, 186, 212

anomalous scattering 65, 67, 70

dispersion 34

scatterers 34, 39

X-ray diffraction 33

Antennapedia gene 326

worm ortholog 328

anterior P lineages 327

antibody

clusters of V, J, D genes 164

diversity 164

genes 164, 165

protein 119

sequences 166

specificity 64

anti-cancer drug cisplatin 306, 308, 309

axostyle 285, 289

Bacillus stearothermophilis 147

bacterial conjugation 87

bacterial gene clusters 110, 113

bacteriophage

defective transducing phage φ80dSU+III genomic mRNAs 125

f2 maturation protein 142, 143, 147

f2 phage 142, 148

helical tail of T4 137, 172

lambda, bacteriophage λ 59, 102, 123, 164,258

lambda head protein assembly 101

mRNA 147

R17 phage 161, 163, 164, 178, 179

T4 coordinated regulation 111

T4 one-step growth experiments 112

T4 phage 58–9, 87–8, 110–12, 123–4, 130, 137, 139, 172, 232, 237, 255, 267–8, 287–8

P22 phage 96, 123

T5 phage 123, 130

bacteriorhodopsin 278, 296, 302, 311–12

bananas 193

black bananas 208

barnacle muscle 315, 317

basketweave pattern 72, 76

Battle of Hastings 152

Beatles, The 72, 81, 295

Beevers-Lipson strips 2, 23

Bell Laboratories 42, 44, 45, 47

Bessel separation program 322

BILDER 98

bioluminescence 218

Bio-X, Stanford 227

brain energetics 47, 48, 49

Bretscher 16th-century cottage 181

Burkitt’s lymphoma 165, 168

C. elegans see nematode worm

cafeteria see canteen

Caius College 180

High Table 180

playing field 213

‘call me Max’ 183, 191

Cambridge market 265

Cambridge Morris Dancers 60

Cambridge University Musical Society 161

cancer genetics 165, 166

canteen 213, 245, 264, 265, 299, 308, 309, 316, 334, 339, 340

celebration party 318

Clark Center at Stanford 227

hearing about latest breakthroughs 192

hospital canteen 259

long rectangular tables 227

unforgettable experience 192

capsomers 138

catalysis

aspartic acid in chymotrypsin 145

catalytic domain of myosin, 332, 333, 334

enzyme mechanism 37, 54, 177, 184, 271, 274, 276, 277, 281

GPDH 194

induced fit 185

RNA catalysis 229

Cell Biology Division 252

cell lineages 268, 323, 325, 327, 328

Central Dogma 185

chain termination 43, 87, 88, 89, 90, 92, 124, 258

alternative termination signal 88

Chaos chaos amoeba 198, 199, 201

chromatin 98, 207

SV40 replication 195

chromosome segregation 284

Churchill College 25, 208, 245, 265, 293, 306, 307, 331, 335, 336

chymotrypsin

α-chymotrypsin 63–70

2Å resolution data 64

2Å resolution electron density 68

dioxan 64

NMR measurements of fast step 271

circular dichroism 80, 82

cisplatin, anti-cancer drug 306, 308, 309

‘co-adaptation’ 111

co-adapted genes 11, 112

colinearity 54, 58

between genes and proteins 255

Cold Spring Harbor 214, 262, 267, 268

1963 Symposium 61, 91

1964 Symposium 113

1965 phage meetings 123

1966 Symposium 92, 131, 141, 142, 162

1969 Symposium 148

1970 Symposium 164

1971 Symposium 37, 48

1979 Symposium 61

college membership 180

complementation

amber mutants 110–12

groups in nematode 255

T4 112

computational enzymology 280

computational structural biology 204, 205, 206, 210, 281

computing

Argus 137

crystallographic computing 23

EDSAC I computer 5, 13, 27

EDSAC II computer 5, 6, 11, 13, 27, 29, 30

eigenvalue-filtering routine 95

IBM 7090 computer 52, 53, 137

Remington Rand UNIVAC 1103A computer 2, 23

VAX-11/780 computer 320

consistent force field (CFF) 272, 280

constant discussions 183

countercurrent distribution (CDD) 126, 128

Covent Garden 329, 334

‘Crick Week’ 58, 184, 185, 253

Crick’s adaptor hypothesis 162

Crick’s Central Dogma 185, 186, 262

culture

blueprint for scientific research 171

creative research at the bench 218

critical discussion 119

democratic participation 192

eliminate distinctions of class 99

idea ventriloquist, Aaron Klug 207

incredibly stimulating environment 194

interdisciplinary 21

interdisciplinary approaches 219, 222

interdisciplinary exchange 227

spontaneously interdisciplinary 213

tea breaks 218, 274, 280

cytoplasmic streaming 198, 201

Dansyl-Edman 117

decorated actin filaments 173, 220, 222

arrowheads 200, 201

chevrons 77

decorated F-actin 173

decoration 200, 201

developmental biology 194, 195, 229, 238, 252, 253, 287, 323, 324, 328

developmental timing 324

localised positional cues 324

mammalian development 189

diagonal method 151

Dictyostelium 216, 222, 223, 225, 228, 247, 250, 287, 289

dielectric constant 275, 277

difference Fourier method 52, 252

difference maps 53–4, 252, 312

diffractometer

optical diffractometer 95, 136, 245

X-ray diffractometer 33, 64, 137, 186, 265

diisopropylfluorophosphate (DFP) 65

direct-space averaging methods 96

DNA nucleotide precursors 258

DNA sequencing 119, 150, 163, 195, 253, 254, 257, 258, 259, 264, 308

mab-5 sequence

operator of phage lambda 258

phiX174 genome 264

DNA-histone cross-links 308

Drosophila 267, 326, 329

downstream genes 90, 111, 141, 144

dynein 284, 285, 287, 288, 289

cytoplasmic dynein 287

heavy chains 287

E. coli 47, 58, 87, 88, 91, 92, 101, 110, 124, 125, 126, 127, 128, 129, 130, 131, 141, 142, 143, 147, 159, 170, 179, 187, 219, 233, 237, 241, 268, 287, 292

5S RNA 127

enzymology of E. coli tRNA 237

EDSAC I computer 5, 13, 27

EDSAC II computer 5, 6, 11, 13, 27, 29, 30

electron crystallography 293, 296, 301

electron microscopy

courses in high resolution EM 284

cryo-electron micrographs 246

cryo-electron microscope, microscopy 205, 321

diamond knife 74, 75

filtered electron micrograph 136

low dose EM methods 246, 263

Philips 300 electron microscope 263

Philips CM-120 321

Phillips microscope 173

Electronic Delay Storage Automatic Calculator see EDSAC

electrophoresis

2D paper 117, 120, 143, 151, 159, 315

acrylamide gel 130

equipment 161

flat polyacrylamide gels 163

high voltage electrophoresis 143, 164

RNA sequencing using 127

peptide separation 212

slab-gel 245

electrostatic environment 274

electrostatic model 276, 277

electromagnetic theory 275

electrostatic preorganization 276

energy transduction 218, 219, 222, 227

English National Opera 335

enzymatic reactions 271, 274, 281

enzyme-substrate complex 94, 274

enzyme 54, 143, 162, 232, 236, 239, 271–81

DNase I 263

elastase 151

GPDH 117

heme-enzymes 51, 52

hexokinase 185, 186

lysozyme 37, 40, 54, 55, 67, 68, 70, 94, 95, 120, 121, 205, 273, 274, 276

motor enzymes 284, 286, 289

protein disulfide bond formation 91

restriction enzyme 150, 258

ribitol dehydrogenase 212

RNase P 130, 237, 238, 241

steric strain in lysozyme 264

enzymology

of E. coli tRNA 143, 237

of tRNA processing 237, 238

eukaryotes 164, 184, 237, 247

diploid 111

Physarum 59

eukaryotic chaperonin 206

eukaryotic organisms 267

EMBL 299, 301, 302, 321

European Molecular Biology Organization (EMBO) 191, 274, 299, 302

Evensong 60, 329

EXAFS 45, 46

Fo-ATPase 280

F1-ATPase 246, 280

faculty position 88, 94, 144, 174, 186, 214, 219, 262

ferromagnetic impurities 42, 44

flight muscle 71–8

flying in gliders 217

Folding Club 254

folding 83, 92, 256

folding problem 254, 273–4, 280

folding project 273

native-denatured transitions 80

footprinting 122, 308

formyl-methionine residue 141, 162

N-formyl methionyl-tRNA 142, 147, 158, 162, 212

N-formyl [35S] methionyl-tRNA 143

Fortran 137, 139, 140

Fortran for the hopelessly ignorant 172

FORTRAN program 137, 245

poetry 96

Fourier synthesis program 27

Fourier transforms 136, 285, 298, 245, 300

3D Fourier transform 139, 140

Fourier Transforms and X-ray Diffraction 172

frameshift experiment 59, 90

freezing specimens

freeze fracturing 302, 332, 333

liquid propane 333

machine 333, 336

French School 111

Fresnel images 139

FRODO 98

frozen-hydrated 321, 322

gene duplication 34

genetic code 34, 42, 54, 58, 59, 89, 94, 141, 158, 162, 163, 230

genetic techniques 91

giant waterbugs 71, 77

glutamate neurotransmitter 47, 48

golden age of molecular biology 154

GPDH 117, 121, 122, 194

gradient of polarity 90

mathematical gradient model 329

morphogenetic gradient 324

graduate student 23, 24, 25, 29, 34, 58, 83, 87, 93, 100, 102, 116, 117, 119, 121, 122, 123, 124, 147, 152, 153, 174, 178, 183, 184, 190, 193, 195, 214, 218, 223, 226, 237, 244, 246, 249, 253, 263, 266, 267, 283, 294, 307, 308, 326, 330

haemoglobin x, xi, 3, 4, 7, 9, 16, 23–40, 44, 46, 48, 148, 308

α and β polypeptide chains 31

absolute hand 34

allosteric mechanism 252, 279, 292

chain tracing 32

correct positions of heavy atoms 27

globin fold 32, 34

heme-heme interactions 44–6

high spin structure 279, 280

laevorotatory amino acids 34

Monod model 45–6

Perutz’s 1970 mechanism 46

planar low spin structure 279

salt bridge constraints 45, 46, 47

Harker phase-circle diagram 28, 29

Harker’s isomorphous replacement methods 9

heavy-atom derivatives 4, 5, 8, 9, 10, 25–30, 64, 65, 67, 95, 117, 186

heavy-atom positions 25, 307

Helen Hay Whitney Foundation 51, 67, 189

helical reconstructions 173, 220

helix-coil transition 80

heterozygosity 111

hexokinase 185, 186

diffraction to 2Å resolution 186

HHMI grant 321

Hirsch Index 84

Hirschegg 208

Holmes-Huxley mirror-monochromator 94, 317

homologous recombination 222, 228

host-induced modification 58, 59

Hut, The 3, 24, 25, 26, 42, 267

hydrophobicity 339

hypochromicity 80

IBM

IBM 360/44 at Institute of Theoretical Astronomy 172, 173

IBM 407 punched card machine 2

IBM 650 magnetic drum computer 2

IBM 7090 at Aldermaston 53

IBM 7090 in London 137

IBM 7070 near Soho 52

IBM cards 172

IBM computer 63

IBM in Tokyo 245

IBM punched cards 65

IBM OS360 96

Hollerith cards

icosahedral viruses 95, 100

icosahedron 1

image analysis 136, 244, 245, 247, 250, 285

immunogenetics 166

induced fit 185

initiation of protein synthesis 141, 142, 143, 144, 146, 147, 151, 162, 178, 212

initiation complexes 179, 206

initiator 159, 162

AUG 178, 179

AUG codon 144

methionine, methionyl tRNA 142, 147

regions 179

Institut de Biologie Moléculaire, Geneva 154, 163, 194

in vitro motility assay 216, 222–6

in vitro reconstitution 219

isomorphous 51

compounds 28

derivative 52, 54, 194

replacement 8, 9, 21, 25, 26, 33, 34, 35, 39, 59, 67, 70

IVIC, Venezuela 315–22

Jesus College 252, 256

Journal of Molecular Biology 55, 58, 69, 124, 178, 207, 252, 315, 320, 322

Editor-in-Chief, Kendrew 191

Joyce-Loebl microdensitomer 5, 52

kinesin 233, 238

King’s College, Cambridge 60, 75, 80, 99, 113, 120, 121, 122, 154, 161, 181, 186, 203, 217, 253, 264, 268

‘Bull Sessions’ 253, 264

High Table 113, 268

King’s College, London xii, 292, 293, 295

Koshland model 44, 45, 185

Lac

operator 86, 90, 91

operon 86, 87, 88, 90, 92

repressor 162, 187

lactate dehydrogenase 37, 122, 213

Lake Como training course 69

laser tweezers 83

leukaemia 237, 312

avian leukaemia 165

oncogene 165

viruses 165

Levinthal paradox 274

light-harvesting complex, LHC-II 301, 302

lineal (intrinsic) cues 326

lizards, hibernating 296, 297

lobster muscle 117, 122, 194

lysozyme see enzyme, lysozyme

magnetic resonance spectroscopy 44, 47

NMR magnets 83

non-invasive NMR methods 47–8

Marine Biology Laboratory (MBL) 218

Mathematics Laboratory, Cambridge 26

maximum likelihood method 300

Mecca 262, 300

mechanical workshops 81

membrane proteins 91, 92, 103, 148, 263, 296, 301, 302

mercury heavy atom derivatives

mercury atoms 7

mercury diamine (HgAm2) 5

para-chloromercuribenzene sulphonate 5

PCMB 28

mercury labelling of DNA 307

messenger RNA (mRNA) 58, 83, 103, 110, 142, 148, 151, 162, 163, 164, 170

methionine, normal initiator 147

methionine tRNA 158, 162

Met tRNA 159

Met tRNAM, tRNAmet 143, 159

MetM tRNA 159

metarhodopsin 278

microtubules 198, 228, 283, 288, 289

helical MT array 284

Physarum 59

MT polarity 284–6

Ministry of Defence, Kendrew 52, 191

mirror-monochromator camera 94, 317

mitotic mutants 286

molecular genetics 99, 151, 158, 166, 167, 219

Molecular Genetics Division 178, 179, 192, 193, 194

molecular dynamics 122, 277–8

microscopic electrostatic studies 280

multiscale modelling 276

Moiré patterns 139

Molecular Genetics Division 136, 158, 178, 192

molecular mechanics 272, 276

Cartesian coordinate description 272

Cartesian force field programs 272

molecular mechanism

anti-cancer drug cisplatin 306

muscle 222, 226

molecular motions and flexibility 205

normal modes 206

molecular replacement 34, 64–5

Molteno Institute 4, 52

morphogenetic gradients 106, 286, 324, 329

multi-cellular

assembly 101

organisms 47

muscle 26, 47, 54, 71–8, 117, 171, 172, 173, 194, 198, 199, 200, 201, 215, 218, 219, 220, 222, 224, 227, 243, 247, 249, 262, 263, 264, 265, 285, 315–22, 331, 332

live muscle tissue x, 94, 219

molecular architecture of muscle filaments 54

non-muscle 222, 255, 263, 322

worm muscle 251, 254, 255, 256, 269, 327

Muscle Club 318,

Musical Club, Cambridge University 118

mutagenesis 59

alkylating mutagens 59

C. elegans 161

ENU, mutagenic mechanism 59

hydroxylamine 234

myofibril structure 72

myoglobin 1–21, 24, 26, 33, 36, 37, 44, 51, 63, 64, 68, 69, 94, 95, 263

1.4Å electron density map 19, 52

2Å resolution map 13, 30, 34

2Å myoglobin map 12, 30

6Å resolution 10, 18, 28, 32

9,600 reflections 13

azide complex 52, 54, 55

azide-ferrimyoglobin 252

bound sulfate ion in azide complex

deoxymyoglobin 54

distal histidine residue

forest of steel rods 17

high resolution 11

horse myoglobin 4

metmyoglobin 52, 53, 54, 55

oxymyoglobin

similarity to haemoglobin 31

sorting board 35

sperm-whale myoglobin 4, 17

myosin 72, 171, 172, 199, 215–28, 243, 245, 246, 247, 248, 254, 255, 256, 262, 285, 317, 319, 321, 322, 324, 331–6

coated beads 216

concentration 220, 221

crossbridge xi, 71, 77, 174, 285, 317, 331, 332

Dictyostelium myosin 222

head-head interaction 322

heavy chain genes 255

heavy chain multiplicity 255

heavy meromyosin 201, 202

high-risk attempt to visualise cross-bridges 331

interacting-heads motif 321, 322

light chain domain 219, 333, 334

light chain kinase 322

myosin I

myosin light chain kinase 322

myosin step size 226

non-muscle 222, 255, 263, 322

Physarum myosin 201

scallop myosin 219

subfragment 1, HMMS1 200, 220, 225, 228, 250, 333, 337

myosin-like motor 222, 228

myxobacteria 101, 102

NASA, Scientist-Astronauts 153

nascent protein 83

near-atomic resolution 3, 25, 296, 302

negative staining 95, 99, 174, 198, 199, 200, 244, 262, 284, 285, 298, 319

negatively stained, various structures 95, 96, 173, 187, 199, 220, 295, 321

nematode worm 89, 239, 242, 252, 269, 286

canonical alleles 255

C. elegans 59, 130, 152, 161, 242, 255, 256, 264, 267, 269, 286, 289, 323, 324, 325, 328

cell lineages 268, 323, 325, 327, 328

coelomocytes 327

development 288

embryos 287

genetic analysis of 268

histology of 239

mab-5 mutant 325–8

nervous system 286

Worm Breeder’s Gazette 269

nepotism rules 144

neurotransmitter 47, 48

neutron scattering 312

Newnham Croft School 306

nicotinic acetylcholine receptor 296

Nitella 222–3

NMR see magnetic resonance

Nobel Prize ix, xii, 3, 23, 60, 74, 80, 115, 117, 119, 102, 162–3, 189, 206, 208, 229, 292–3, 299, 318, 323, 325

nonsense codons 43, 54, 124, 125, 141, 142, 231

nucleosomes 96, 239, 306, 308

platinum binding 306

ochre 88, 89, 91, 94, 124, 233, 234

mutants 88, 91

stop codons 94

suppressors 89, 124, 233, 234

tRNA mutant 234

opal suppressors 124

operon 104, 111, 141, 219

expression 86, 90

Lac 86–8, 90

operons 88, 110, 241

optical diffractometer 95, 136, 245

optical diffractometry 77, 95, 200, 244, 249, 285

optical filtering 136, 139, 284

optical properties of nucleic acids and proteins 80

overlapping genes 264

peptide bond hydrolysis 184

Perse School for Girls 336

Peterhouse 13, 18, 55, 96, 99, 133, 135, 160, 191, 293, 299, 305, 306

High Table 294

phage RNA polymerase 143

phases 7, 8, 13, 26, 33, 52, 98, 136, 137, 138

best phase 9, 11, 12, 29

phase-angle probability 9, 11

phase combination 34

phase determination 9–12, 28, 34, 38, 67, 96–7

phase determination program 67

phase problem 6, 24–5, 137

‘rocket’ for phase determination 54

phage genetics 89, 109, 112, 113

phosphate dissociation 332

photographic data collection 64, 65, 66

photoisomerization 278, 181

photosynthesis 278, 281, 302

Physarum 59, 197, 222

Physarum actin 197–202

Physarum myosin 201, 202

Physarum polycephalum 57, 198, 199, 202, 222

platinum binding 306

polar effects 111, 141, 144, 148

polar mutants 90

polarisable dipoles 275

polygenic messenger RNA 142

polymerase structures 186

polynucleotides 80

polypeptide chain initiation 141, 142, 146, 147, 148, 159

polypurine stretches 179

position-dependent score in sequence recognition 340

positional information in worm 323, 328

posterior P lineages 327

potential energy function 205

power stroke 77

Princeton 86, 116, 181, 287

programmable algorithm 29

prokaryotes 164, 184

promoter mutations 90, 92

Protein and Nucleic Acids Division 178, 179, 192

Protein Chemistry Division 119, 120

protein crystallography 55, 70, 115, 120, 177, 189, 190, 195, 293

end of an era 55

protein dielectric constants 277

protein energetics 339

protein folding see folding

protein kinase C 322

protein kinase (PKA) 211–14

dynamic molecular switches 214

protein kinase structures 212, 214

protein secretion 91, 92

protein stabilities 277

punched paper tape 5, 29, 137

purified polycistronic messenger 142

pyrrole nitrogens 53

QM/MM 274, 276, 277, 281

quantitative in vitro motility assays 216, 218, 222, 223, 224, 225

quantum mechanics 271

hybrid orbitals 273

low dielectric environment 276

quantum Hamiltonian 276

quantum mechanical model 273

quantum mechanical wave functions 271

wave function 271, 272, 277

quasi-atomic model of F-actin 248

quasi-equivalence 100, 138

quaternary structure of haemoglobin 44, 45, 46, 47

radioactivity assays 128, 238, 306, 332

recognition sequence 259

recombinant DNA, revolution 164, 253, 255

redox potentials 277, 279

reorganisation energy 276

replication control 59

Replicon Model 59

repressor 86, 98, 162, 187, 258

repressor dimer 259

restriction enzymes 150, 258

rhodamine phalloidin 224

rhodopsin 278

ribitol dehydrogenase 212

ribosome 38, 83, 103, 121, 122, 141, 144, 147, 151, 159, 161, 170, 171, 174, 175, 186

5S ribosomal RNA 150, 159, 162

30S ribosomal subunit 130, 131, 147, 186, 237

50S ribosomal subunit 186

70S ribosome 122

lizard ribosomes 291–301

ribosome crystallography 122, 296, 297, 298

ribosome profiling 177–81

ribosome-protected mRNA 179

ribozymes 38, 83

Richards Box 98, 214

right-handed helix 34, 80

RNA

5S ribosomal RNA 150, 159, 162

bacteriophage f2 147, 148

catalytic subunit RNAse P 238

extra-stable tetraloops 83

fingerprinting methods 258

makes protein, Central Dogma 186

polymerase 142, 143, 147, 186, 259

processing 229, 237

RNA diagonal work 151

RNase P 130, 236, 237, 238, 241

secondary structure 144, 145, 147

sequencing 120, 127, 163, 164, 178, 179, 212

RNA Society 130

RNase P 130, 236, 237, 238, 241

RNase T1 oligonucleotide 151

role models, none for women 181

Rossmann Fold 122

rubredoxin 46, 49

sabbatical in Heidelberg, Reedy 78

Sanger fingerprint 179

Sanger sequencing technique 162, 258

scientific exchange 181

Scientist-Astronauts in NASA 153

separating peptides 212

sequencing of LDH 214

sequencing RNA xii, 120, 212

sewing machine 81

Siemens Elmiskop IA 76, 173, 220

single anomalous dispersion (SID) 34

single isomorphous replacement (SIR) 34, 39

six-hydroxyl group of glucose 185

sliding filament model xi, 262, 284

slime mould 102, 199, 201, 202, 222, 228

‘small square’ lattice in Z-bands 76, 77

solid tumours 309

solvent content of protein crystals 68, 69, 70

somatic division lineage 59

specific tRNAs 141

spliceosome 130

square lattice in I-bands 72

‘stability numbers’ in RNA double strand formation 82

Stanford provost, Condoleezza Rice 227

statistical mechanics 271

steric block hypothesis 246, 263

steric blocking mechanism 222

steric blocking model 263

stochastic simulation in molecular mechanisms 206

stop codons 94, 124, 255

structural biology 34, 36, 37, 38, 55, 69, 151, 161, 169, 170, 171, 173, 174, 175, 183, 184, 187, 194, 203, 204, 205, 206, 210, 217, 219, 222, 223, 227, 262, 264, 265, 278, 280, 281, 291, 295, 300, 301, 315, 321, 322

Structural Studies Division 54, 136, 161, 177, 189, 204, 206, 264, 293, 300, 318

Su3+ suppressor tRNAtyr 152

substrate analogues 183

substrate specificity 121, 184

sucrose gradient 151, 179

swinging crossbridge 219, 225, 226

Swiss cheese and banana 181

symmetric sequences, twofold 259

synchrotron 38, 250

Daresbury synchrotron 248

DESY synchrotron 317

synchrotron research 316

synchrotron X-rays 78

tarantula thick filaments 320, 321, 322

tarantula muscle 319, 321

teaching

online classes 210

Woods Hole 215

Technion 271

temperature-sensitive mutant 110–11, 142, 237, 256, 267–8

Tetrahymena 247

tetrakis (acetoximercuri) methane (TAMM) 158, 307, 308

‘The Double Helix’ 21, 58, 59, 61, 153, 161, 162, 244, 284

thermally setting clay 32–3

‘threading’ 340

three-dimensional 133–40

data 52

difference map 53

electron density map 13, 67

fold 122

Fourier synthesis 5

image reconstruction xi, 244, 250, 308

reconstruction 136–40, 319–22

structure 25, 36, 51

tobacco mosaic virus (TMV) xi, 93, 136, 158, 244, 300, 315

Towering Heroes 205, 206, 210

transcription 110, 206, 229, 259

direction 112

initiation 86, 88, 90

reverse transcription 154

site of initiation 88

termination 88

transducin 278

transfer RNA (tRNA)

acridine-induced mutants 231, 232, 235

anticodon 124, 128, 131, 152, 167, 185, 231, 234, 236

crystal structures 239

E. coli tRNA processing 143, 237

formyl-methionine tRNA 162, 212

genes with mutated anticodons 124

mature tRNAs 130, 235

methionine tRNA 158, 162

Met tRNAM 143, 159

ochre-suppressing tRNA mutant 233, 234

precursor tRNAs 130, 235, 236, 237

specific tRNAs 141

Su3+ suppressor tRNAtyr 152

Su+ tRNA 127

Su– tRNA 128

tRNA processing 237

tRNA synthetases 141, 142, 185, 186, 212

transition state in enzymes 274, 276, 305

transition G-C to A-T 255

triplet code xi, 42, 43, 58, 59

Trinity College 96, 217, 293, 311

High Table 96

tropomyosin-troponin 221, 222, 244, 247, 248, 249

complexes 219, 220, 227, 228, 245, 246

troponin 244, 245

absence of Ca2+ 220–22, 245–7

Ca2+-sensitive binding 262

calcium-binding subunit (TnC) 245

tubulin 59, 285, 286, 289, 308

affinity columns 286

exogenous tubulin 285

turnip crinkle virus (TCV) 93, 99

twinning 64

eliminated twinning 64

twinned chymotrypsin crystals 64

two-dimensional crystals 296, 301, 302

two-dimensional fingerprinting 117, 120, 127, 143, 151, 234, 258

UAA 89, 124

UAG 89, 124, 128

UGA 124, 152

unc-45 256

unc-54 255

unidirectional movement 233

universal polypeptide initiator 162

University California, Berkeley 18, 79, 81, 85, 121, 144, 181, 298

uranyl acetate xi, 198, 200, 245, 298

US National Academy 154, 166, 184, 192

vesicular stomatitis virus 146, 148

vibrational modes 272

viral synthetase 179

virus xi, 38, 54, 93, 97, 98, 99, 100, 110, 136, 142, 172, 193, 236, 257, 267, 284, 300, 315

DNA viruses 100

human wart virus 95, 138

λ assembly pathway 100

leukaemia virus 165

tomato bushy stunt 94, 95

turnip crinkle 93, 99

Walter and Eliza Hall Institute 157, 164, 166

‘wandering spot’ methods 257, 258

Weizmann Institute 204, 209, 271, 272, 273

wine 55, 163, 180, 206, 207, 294, 295

best cellars in Cambridge 294

wire models 14, 94

Woods Hole 94, 216, 218, 219, 223, 268, 316, 317

worm see nematode worm

Worm Breeder’s Gazette 269

X-ray 1–13, 71–8

crystallography 36, 46, 93, 99, 137, 161, 183, 248, 308

diffraction 33, 38, 94, 97, 100, 172, 194, 215, 246, 252, 293, 315, 317, 320

generator 25, 29, 52, 96, 317

microfocus generator 94

photograph, film 4, 38, 65, 95, 143, 179

reflections 317

scattering 219, 247, 250, 311

X-ray diffraction patterns 246, 293, 317, 319

X-ray fiber diffraction 77, 332

Yale 41, 47, 144, 169, 174, 177, 181, 183, 185, 186, 212, 229, 237, 238, 241, 301, 311, 331

Z-bands 71–7

filament torque expression 72

zinc finger 307

zona pellucida (ZP) 195, 196