- Email: [email protected]
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˚ . Since then, there have phases were extended all the way from 6 to 3.5 A ˚ been a number of examples where phases have been extended to 3.5 A 24 using merely an initial hollow shell model for a virus to initialize phases ˚ resolution. to about 20 A Conclusion
The reader may have noticed that many of the concepts we now take for granted were initially met with opposition. It is satisfying to see so many of the originally contentious suggestions being now fully accepted. Nevertheless, even now my colleagues and I have had publication difficulties with a paper containing novel methods and unexpected results.25 Acknowledgments A great deal of history is covered in this personal review. It would be impossible to acknowledge all the individuals who have influenced and encouraged me over the past 50 years. I thank all those many individuals who have guided me through problems to a great many successes. Mostly, I have had a lot of fun. Hopefully, I have been able to help others to enjoy their scientific endeavors. One of the greatest rewards has been to have found lasting friendships in almost every part of the world. I thank primarily the NIH, NSF, and Purdue University for continuing financial support.
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J. Tsao, M. S. Chapman, M. Agbandje, W. Keller, K. Smith, H. Wu, M. Luo, T. J. Smith, M. G. Rossmann, R. W. Compans, and C. R. Parrish, Science 251, 1456 (1991). 25 R. J. Kuhn, W. Zhang, M. G. Rossmann, S. V. Pletnev, J. Corver, E. Lenches, C. T. Jones, S. Mukhopadhyay, P. R. Chipman, E. G. Strauss, T. S. Baker, and J. H. Strauss, Cell 105, 127 (2001).
[3] Personal X-ray Reflections By U. W. Arndt One of the results of having been around for quite a long time and of having met many of the great figures of the past is that one gets asked to write historical surveys of one’s subject. To do so properly is not at all easy: according to Horace Judson, author of the majestic history of Molecular Biology, ‘‘The Eighth Day of Creation,’’ practicing scientists are poor witnesses. They like to look back on their own work as though it had all conformed to a well-thought out plan in which each advance followed from the previous one, itself, of course, preferably made by themselves or their pupils.
METHODS IN ENZYMOLOGY, VOL. 368
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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In practice, of course, progress has often depended on luck and on the timeliness of the project, that is, on the availability of the necessary tools and techniques, and on the political atmosphere that encourages funding of the field. Accordingly, I shall not attempt to write a serious historical survey along the lines of that by Brian Matthews1 in an earlier volume of this series. I shall, instead, merely try to record my reflections on some of the events I was fortunate enough to witness or be involved in myself. Much of my working life has been spent in Cambridge and so my account is naturally biased toward what happened there. I began working in crystallography (but not in protein crystallography) in 1944 in the Cavendish Laboratory Cambridge, where Max Perutz had already started on his self-imposed task of determining the crystal structure of hemoglobin. At that time many small-molecular crystallographers thought that this was an insoluble problem: Even if by some miracle he managed to measure the intensity of tens of thousands of X-ray reflections, and even if by some further miracle the phases of those reflections were revealed to him, it would take 20 years of churning away on the mechanical calculating machines of the day to calculate the electron density in order to plot three-dimensional maps of the molecule. Besides, there was no guarantee that the molecule, which, after all, reacted only when in solution, would have the same shape when in that state as when in a solid crystal. I once asked Max Perutz whether at that time he had an inkling that computers were just around the corner. He told me, no, he just carried on on his chosen path, hopeful that each problem would be solved in turn. The world acknowledges a project as having been successful only when it is conducted to a conclusion. In his Nobel Prize lecture in 19622 Perutz quoted Sir Francis Drake’s famous prayer . . . . ‘‘Grant us also to know that it is not the beginning but the continuing [of any great matter] until it is thoroughly finished which yieldeth the true glory.’’ This is an excellent maxim except in those cases when one is hitting one’s head against a brick wall. The mark of successful researchers is that they recognize the brick wall for what it is and that they then switch to a more promising problem.3 Mere ideas are ten-a-penny, as Ken Holmes was fond of saying. This is particularly worth remembering today when what might be a solution is arrived at by guess-work, now called computer simulation, and when the real answer must be checked by practical experiment. I am reminded of an example in electron microscopy. 1
B. W. Matthews, Methods Enzymol. 276, 3 (1997). M. F. Perutz, Nobel Prize Lecture, Stockholm (1962). 3 A. D. McLachlan, Personal Communication. 2
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At a conference on electron microscopy in 19704 Walter Hoppe of Munich presented a minutely argued treatise on the benefits in resolution and contrast which could be expected by placing a mask in the direct electron beam, provided this opaque mask was exactly centered. The next speaker was Nigel Unwin who showed actual examples of the improvements which he had achieved by the much simpler procedure of mounting a gold-coated spider’s thread at the exit of the objective lens. ‘‘But how did you succeed in exactly centering the thread?’’ Hoppe wanted to know. The answer, ‘‘Oh, we just made the spider walk across the hole,’’ led to a collapse of the audience in laughter. Poor Walter Hoppe! He was a very nice man as well as being a brilliant scientist who made important contributions in many fields ranging from instrument development and X-ray optics to phase determination, but he tended to be caught on the wrong foot at conferences. There was the occasion when he commented on a talk by Caroline MacGillavry, whose name he mispronounced Macgilla´ vry several times. She did not correct him, but in her reply she referred to him only as Professor Hop. But let us return to protein crystallography. I was fortunate in visiting Cambridge from London where I was then working at the Royal Institution, a day or two after Perutz and his team had obtained precession photographs of a crystal of native horse haemoglobin and of an isomorphous crystal of a derivative in which two mercury atoms had been attached to the protein molecule.5 Laying the two photographs over one another with a slight displacement it was immediately obvious that there were intensity differences: in principle, the phase problem had been solved, and the path, though still a long one, lay open for a complete structure determination. Everyone in what was then the MRC Unit was in a state of euphoria and Max Perutz seemed to be floating inches above the floor. X-ray tubes in the early days were not very powerful and exposure times of several days were sometimes necessary. The maximum power that can be dissipated in the tube target without melting it is set by the rate at which heat can be conducted away from the electron focus to the watercooled rear surface of the target. A higher power is possible when the target surface is moved. Starting with those of A. Mu¨ ller,6 several designs of rotating-anode tubes were produced, including Hoppe’s highly sophisticated idea for an anode cooled with liquid gallium or sodium, which has never been developed further. However, although these generators allowed a higher power dissipation in the target, the brilliance of the source 4
Reported in Phil. Trans. R. Soc. Lond. B, 261 (1971). D. W. Green, V. Ingram, and M. F. Perutz, Proc. R. Soc. Lond. A225, 287 (1954). 6 A. Mu¨ ller, Proc. R. Soc. Lond. A125, 507 (1929). 5
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was mostly low, that is, the electron focus on the target was too large to give much improvement in the intensity that reached a small crystal. The 50-kW continuously-pumped rotating-anode tube built by Mu¨ ller at the Royal Institution (Fig. 1) had a foreshortened electron focus several millimeters in diameter; it actually delivered a lower X-ray intensity to a small sample than a 1-kW sealed-off X-ray tube. One of my first acts on starting work at the Royal Institution in 1950 was to recommend the retirement of the monster. We were then able to work in the laboratory without earmuffs to reduce the roar from the 10 horsepower motor and the screech from the stuffing box vacuum seal for the target shaft. Incidentally, the laboratory in which the tube was housed was used by Michael Faraday and in 1950 it still contained some of his apparatus, which looked as though it had not been touched since his days. In the cold winters in the late 1940s Lonsdale and Owston carried on their work in this room: their publication7 contains the statement that the work was carried out at room temperature ( 5 C). Today the room has been restored as the Faraday Museum.
Fig. 1. Mu¨ ller’s rotating-anode X-ray generator at the Royal Institution, London, ca. 1950. The 10-horsepower motor on the right of the bed drove the 600-mm-diameter target via a long shaft running in a vaccum-tight stuffing box. The hexaphase power supply, with water-cooled rectifier valves, was in the cubicle on the left. Vacuum pumps for the tube were under the bench. (Author’s photograph.)
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A compact rotating-anode tube was designed by A. Taylor8 and was further improved by Tony Broad in the MRC Unit in Cambridge.9 Finally, Hugh Huxley and Ken Holmes combined Broad’s anode with a better electron gun based on that of the French Beaudouin tube. This hybrid tube was produced commercially by Elliot Brothers in England, later by GEC, and finally by Enraf Nonius, and was for many years the standard workhorse for protein crystallography. Recently, high-brilliance microsource X-ray tubes have been reintroduced.10 It was well known that a very small electron focus allowed more efficient cooling of the X-ray tube target as the isothermal surfaces in this target became hemispherical rather than planar. Advances in X-ray optics allowed the radiation to be focused on the specimen crystal.11 With suitable X-ray mirrors the focused intensity with a low-power microfocus tube can be made to exceed the intensity in the aperture-limited unfocused beam from a rotating-anode X-ray generator operated at more than 100 times the power. Focusing ellipsoidal and paraboloidal X-ray mirrors had been produced in Prague for X-ray telescopes flown in Russian satellites. My request for reprints brought the literature, accompanied by a puzzled inquiry as to why someone in a laboratory of molecular biology was interested in X-ray astronomy. In due course this led to a fruitful collaboration that resulted in the production of a series of mirrors for use with microfocus tubes.12 It is difficult today to remember the manipulatory skills that were required to mount and align protein crystals on the much cruder X-ray goniometer heads and on the X-ray cameras that lacked some of the modern facilities (Fig. 2), and there was much to learn about the behavior of protein crystals. It seems highly appropriate that it was Francis Crick, who was much given to handwaving (Fig. 3), who discovered that draughts and temperature gradients near a mounted crystal would lead to a distillation of solvent from one part of the specimen tube to another and thus to changes of unit cell parameters.13
7
K. Lonsdale and P. Owston, J. Glaciol. (1948). A. Taylor, J. Sci. Instrum. 26, 225 (1949); Rev. Sci. Instrum. 27, 757 (1956). 9 D. A. G. Broad, Rotating-anode X-ray tube. UK Patent Applications, Nos. 5172, 5173, 12761, 38939, 13376 (1956). 10 U. W. Arndt, J. V. P. Long, and P. Duncomb, J. Appl. Crystallogr. 31, 936 (1998). 11 U. W. Arndt, P. Duncomb, J. V. P. Long, L. Pina, and A. Inneman, J. Appl. Crystallogr. 31, 733 (1998). 12 L. Pina, A. Inneman, R. Hudec, U. W. Arndt, N. Loxley, G. Fraser, M. Taylor, and J. Wall, Proc. SPIE 4144, 165 (2000). 13 F. H. C. Crick and B. S. Magdoff, Acta Crystallogr. 9, 901 (1956). 8
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Fig. 2. Unicam single-crystal X-ray camera in the foreground, with cylindrical film holder beside it. Until about 1955 most protein photographs were taken with this camera (in the background a Philips Debye-Scherrer camera is seen mounted at another window of a vertically mounted sealed-off X-ray tube). (From LMB Archives.)
The real value of protein crystallography started to emerge only when the first structures were carried as far as atomic resolution. Only then did it become apparent that the molecule as seen by the crystallographer had the shape that it took up in solution and that a knowledge of the shape of an enzyme molecule could explain the nature of enzyme–substrate interactions. Lower-resolution maps on which individual atoms could not be identified still contained many ambiguities. Before it was realized that proteins consisted of long folded polypeptide chains the ‘‘cyclol’’ theory of protein structure, which postulated interlocking rings or cages, had some ˚ resolution map of myoglobin had come adherents. By the time the 5-A out the cyclol theory was largely discredited, but I once witnessed Isidore Fankuchen standing in front of this map and, with his tongue in his cheek, pointing out how clearly visible the cyclols were! In the 1960s some people believed that the successes of X-ray crystal structure determination were so spectacular that it would soon become the standard technique for amino acid sequence analysis. This has not
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Fig. 3. Francis Crick, waving his hands (see text). (From LMB Archives and Prof. Uno Lindberg.)
happened, partly, of course, because sequencing techniques have also been greatly improved and automated since Fred Sanger’s heroic efforts in sequencing insulin, for which he was awarded his first Nobel Prize. At the party following his second Nobel Prize in 1980 for base sequencing in DNA several speakers spoke of Fred’s unassuming modesty. After a good deal of champagne had flowed, Fred was forced to reply to his well-wishers, which he did with the following words: ‘‘Everyone says how modest I am; I may be modest, but I am also bloody good.’’ The applause nearly brought the roof down. The solution of the structures of myoglobin by Kendrew and of hemoglobin by Perutz (Fig. 4), followed in 1965 by that of the first enzyme, lysozyme, by David Phillips and his team at the Royal Institution,14 was a great stimulus to look at the structure of other proteins. Sir Lawrence Bragg, the Cavendish professor, had been giving enthusiastic moral support to the hemoglobin group, which became the MRC Unit for the Study of Molecular Structure of Biological Systems in 1947 and, in 1962, the MRC Laboratory of Molecular Biology (LMB). Based on his own earlier pioneer work 14
C. C. F. Blake, D. F. Koenig, G. A. Mair, A. C. T. North, D. C. Phillips, and V. R. Sarma, Nature 206, 757 (1965).
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Fig. 4. Max Perutz (left) and John Kendrew, sketched by W. L. Bragg, ca. 1955. (From LMB Archives.)
on the families of silicate minerals,15 Bragg believed that the elucidation of a few protein structures would reveal the common features of all proteins and thus ‘‘explain the Secret of Life.’’ In this belief he was wrong: different proteins have turned out to be as varied and as fiercely individualistic as their investigators. I should like to express a heretical belief of my own here. Certainly Bragg was interested in the Secret of Life, but I believe that his primary interest in protein structure was different: his career had been a splendid progress from the simplest crystal structure of all, that of sodium chloride, to the structures of metals and minerals. He was now faced with the largest and most complex molecules known to man, and that is what excited him. Most of the early protein crystallographers had started life as physicists or physical chemists and were essentially interested in the structures as structures, and not then as stepping stones to the answering of biological questions. It needed the entry into the field of biochemists, geneticists, and other biologists to ask these questions and to find answers to them. It would be nice to think that all the new instruments and techniques that were developed by physicists and engineers who moved into molecular biology were part of a logical program devised after experimental bottlenecks had been identified. In fact, I suspect that many instrument makers like myself were partly motivated by a desire to continue to play with the Meccano construction sets and model railways of our childhood. It was sometimes difficult to know when to stop perfecting an instrument and to 15
W. L. Bragg, ‘‘The Atomic Structure of Minerals.’’ Cornell University Press, New York, (1937).
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start using it for actual research. When David Phillips and I had built our linear diffractometer16 (Fig. 5), I would have gone on making small improvements when David started to collect data from a myoglobin crystal, in accordance with his oft-repeated saying ‘‘The best is the enemy of the good.’’ The ‘‘Secret of Life’’ made a comeback many years later when the protagonists of new synchrotron radiation sources were looking for scientific allies among biologists. The synchrotron beams were sufficiently intense to offer a promise of investigating structure changes in biological materials dynamically, and, after all, it was the undergoing of changes that distinguished life and biological materials. A questioning of what interesting biological systems there were in which changes should be investigated usually provided the answer: ‘‘well, there is muscle and er . . . er . . . muscle . . . and muscle . . . !’’ Whatever the truth is in these speculations, the search was on for interesting proteins to investigate. The path soon led away from the farmyard: John Kendrew had an early arrangement with the London Zoo that they would notify him of the deaths of unusual diving animals such as penguins, seals, and sea-lions, from whose carcasses he would extract myoglobin. David Blow obtained his cytochrome c samples from tuna fish hearts, but prizes awarded by colleagues would probably have gone to Herman Watson who looked at lobster GPD (glyceraldehyde phosphodehydrogenase). A consignment of fresh West Country lobsters arrived at intervals for processing and for extracting the protein. Fortunately, for a reason not clear to me, the claws were not suitable for this purpose and were thus available for distribution to Herman’s friends. Not all attempts to obtain proteins from raw materials were equally successful. Francis Crick at one time organized a group of us to sit in a circle and breathe in the fumes from freshly cut onions. He collected our tears, but when he came to analyze them they contained very little lysozyme as hoped, but were almost pure onion juice. The most attractive raw materials are not always the most suitable ones for scientific purposes. In 1954 Dennis Riley gave a Royal Institution Friday Evening Discourse17 on the subject ‘‘The Raw Materials of Life.’’18 Several of the lecture demonstrations and experiments involved hen eggs, at that time still rationed in Britain. However, the Egg Marketing Board was sympathetic and assisted Riley in obtaining a supply of particularly large and beautiful eggs for such a high scientific purpose. These 16
U. W. Arndt and D. C. Phillips, Acta Crystallogr. 14, 807 (1961). D. P. Riley, Proc. R. Inst. Gt. Brit. 35, 363 (1952). 18 D. P. Riley and U. W. Arndt, Nature 171, 144 (1953). 17
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Fig. 5. The first manual linear diffractometer, ‘‘P. P. Ewald’s grandchild.’’ (Author’s photograph.)
‘‘Discourses’’ were important social occasions and it was pretty daunting for the lecturer to enter the ‘‘Amphitheatre’’ at 9 o’clock sharp to be faced with rows upon rows of the distinguished audience in full evening dress. The lecturer frequently restored his courage with a stiff double whisky just before his ordeal. Riley’s first demonstration was to break an egg into a bowl of oil to show the separation into yolk and eggwhite. The extra-large egg turned out to have a double yolk, as by a rare chance did the next one. The lecturer was clearly extremely concerned whether he was seeing things and his enormous relief was obvious when he caught murmurs from the audience ‘‘look, another double-yolked egg.’’
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I can remember another time when the source of the material could have been, but was not, of great importance. In 1971 Ken Holmes with his co-workers Rosenbaum and Witz from Heidelberg had built a beamline to exploit the synchrotron radiation from a port of the new German electron synchrotron (DESY) in Hamburg and I had gone there to assist them in getting what was to be the first synchrotron radiation diffraction photograph of a muscle specimen.19 We worked all day to get everything ready for the moment when the beam was switched on for the evening shift. Holmes had a gentleman’s agreement with his former colleague Hugh Huxley, who had developed the mirrors and monochromators and the techniques for examining muscles (Fig. 6), that frog sartorius muscle belonged to Hugh; Ken would concentrate on insect flight muscle. (Such gentleman’s agreements were common in the early days, when it was not ‘‘done’’ to start a structure determination of a protein on which someone else was known to be working.) Accordingly, Holmes and his team brought along a specimen of a waterbeetle flight muscle, which had not been photographed properly before at a conventional X-ray source. With some effort Ken was persuaded that it would be particularly valuable to take one diffraction photograph of a well-known material in order to make quantitative comparisons of the two sources. As evening fell, Gerd Rosenbaum was despatched to go frog-hunting at a neighboring pond in company with the DESY gatekeeper’s son. They failed to catch a frog, but fortunately the insect flight muscle photograph turned out to be a sufficiently good one to make a powerful case for the continued development of synchrotron sources at DESY and elsewhere. Holmes’ synchrotron radiation work had a political as well as a scientific importance. John Kendrew had, for some time, been arguing the case for a European Molecular Biology Laboratory (EMBL), which, like CERN (Centre Europe´ en pour la Recherche Nucle´ aire), would be able to undertake research too expensive for individual national laboratories. He came up against opposing claims that there was really nothing sufficiently expensive in molecular biology to justify an international laboratory. The highcost synchrotron radiation work came at the right moment and EMBL was planned at the outset with a main laboratory at Heidelberg and an outstation at DESY. There was and is another EMBL outstation at Grenoble, France, adjacent to the Institut Laue-Langevin (ILL), to benefit from collaborative biological structure work with neutrons from the high-flux beam reactor at the latter institute. The existence of the administrative machinery at the trinational (French, German, and British) ILL was one of the reasons adduced for building the European Synchrotron Radiation Facility 19
G. Rosenbaum, K. C. Holmes, and J. Witz, Nature 230, 129 (1971).
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Fig. 6. H. E. Huxley, adjusting his muscle camera. (From LMB Archives.)
(ESRF) next door to the ILL instead of in Strasbourg, which would have been the site preferred by many French scientists. The decision led to considerable but unsuccessful agitation in Alsace with car stickers and posters protesting against the ‘‘betrayal’’ of Alsace and its capital Strasbourg in depriving it of the synchrotron. Following the work of Holmes at DESY and of Keith Hodgson and his colleagues at Stanford,20 protein crystallographers were quick to realize the advantages of the X-ray flux, which was many times greater than that from conventional laboratory X-ray sources. Before long they started to 20
J. C. Phillips, A. Wlodawar, M. M. Yewitz, and K. O. Hodgson, Proc. Nat. Acad. Sci. USA 73, 128 (1976).
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appreciate the benefits of the tuneability of synchrotron beam lines, which ˚ X-rays. meant that they were no longer exclusively limited to 1.54-A In due course the success of synchrotron radiation sources in the United States, Germany, the UK, Japan, and France led to the demand for more and more ‘‘protein crystallography (PX)’’ beam lines and thus, to a considerable extent, to the plan for a European Synchrotron Research Facility ‘‘to keep up with the Americans’’ and for a National Synchrotron Light Source (NSLS) and an Advanced Photon Source (APS) in the United States ‘‘to keep up with the Europeans.’’ The growth of synchrotron radiation research is illustrated by the fact that the (2001) Editorial Board of Synchrotron Radiation News has members in synchrotron radiation laboratories in 15 countries, all of which have crystallographers who use synchrotron radiation. Many protein crystallographers now think little of making several multithousand mile trips a year to collect diffraction data at a center where they can find a suitable beam line. More importantly, funding agencies are prepared to finance such trips. Data collection runs are now sometimes carried out in as many hours as the number of weeks necessary in earlier days. The development of these shared resources accelerated the internationalization of what had always been a field in which one’s collaborators came from many different countries. One of the privileges of having worked in the field of molecular biology is that there is virtually no country in the six continents in which one cannot find close colleagues and good friends. With the projected zero-gravity protein crystallization and protein structure determination program in the International Space Laboratory such colleagues may soon not be restricted to the earth’s surface. Protein crystallography had first taken off in Britain, where for a surprisingly long time nearly all crystallographers could trace their scientific descent from either W. H. or W. L. Bragg. The explosive growth of the subject in the early and mid-1950s owed much to overseas visitors and postdoctoral workers, especially those from the United States. At the celebration of the twenty-fifth anniversary of the Nobel Prizes to Watson, Crick, Perutz, and Kendrew, which was also the twenty-fifth birthday of the MRC Laboratory of Molecular Biology in Cambridge, and the fortieth birthday of the original MRC Unit, Jim Watson noted that a high proportion of major American professorships of Molecular Biology were filled by alumni of the laboratory. Today news is sent to overseas collaborators by e-mail so quickly that it is often not read before being replied to. In the early days news traveled almost as fast by word of mouth. In 1976 a committee set up in Britain to investigate the efficiency of government-financed laboratories noted that
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the MRC Laboratory of Molecular Biology had an unusually small bill for textbooks and journals. The explanation that ‘‘when someone elsewhere had done something interesting he usually came to Cambridge to tell us about it’’ was received with skepticism. However, this was one of the facts that made for particularly lively seminars with hard-hitting discussions. Sydney Brenner’s not-so sotto voce comments often enlivened a colloquium; Francis Crick, just back from a visit to Leslie Orgel at La Jolla, once reported on Leslie’s latest ideas there that life had started in the primeval soup as chips of apatite and was interrupted by the remark ‘‘I see, chips before fish.’’ Chips also had another influence on molecular biology. Protein crystallography was transformed by the advent of electronic computing; it was arguably one of the first scientific fields that made constant use of computers. Of course, computers could be developed only when the whole field of electronics had advanced sufficiently and, in particular, they needed the introduction of transistors and integrated solid-state circuitry. The development is still far from complete: Ge´ rard Bricogne once described human beings as ‘‘the catalyst in the transition from carbon-based to silicon-based intelligence.’’21 It is difficult to appreciate just how great the developments in electronics and in computing have been. Strangely, while our laboratories had excellent mechanical workshops and instrument makers, it was not for many years that adequate space and manpower were devoted to electronic design and construction. In 1944 there was not a single ‘‘valve’’ (electronic vacuum tube) in the crystallographic laboratory of the Cavendish Laboratory, Cambridge. Fourier syntheses were carried out with the help of Beevers–Lipson strips22 (already a great advance on having to look up sines and cosines in books of tables), and only the senior research students had easy access to motor-driven adding machines. The rest of us had to content ourselves with the slower manual adding machines. The motor-driven calculators had another advantage, discovered by Alex Stokes: when the darkroom clock broke down Alex worked out an appropriate long division so that the silence at the end of its labors signaled the end of the development period. In 1945 J. D. Bernal spoke at a British Institute of Physics Conference on the ‘‘The Future of X-ray Analysis.’’23 He said ‘‘there must be a great development of analytic methods and means of calculation. We are only at the beginning of mechanical and optical methods, and their 21
G. Bricogne, Personal communication. C. A. Beevers and H. Lipson, Proc. Phys. Soc. Lond. 48, 772 (1936). 23 J. D. Bernal, Nature 155, 713 (1945). 22
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improvement is very necessary as calculations are becoming a bottleneck. There is no use in being able to take (X-ray) photographs in a few seconds if calculations take months.’’ The progress in electronic computers depended on the progress of electronics as such (Fig. 7). In about 1945 when I started to develop my own X-ray Geiger counters I had, of course, to build my own electronic amplifiers and counters. Manhattan project electronic designs24 had not yet been declassified, and ‘‘counters’’ were called ‘‘scalers’’ because they
Fig. 7. Construction of XRAC. Electronic instruments were usually assembled from interconnected 19-in. chasses like the ones illustrated here. Today such a chassis, filled with dozens of tubes, could be replaced by one integrated circuit. (From Pennsylvania State College.)
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scaled down the counting rate by 32 or 64 to a rate with which Post Office telephone message registers could cope. (Some years later I started playing with energy-sensitive proportional counters and needed a pulseheight-analyzer; this was known as a ‘‘Kick Sorter’’ because such a device had been built at the Cavendish in which the pulses from the radiation detector were amplified and fed into a loud-speaker coil where they served to catapult —‘‘kick’’ — small steel balls a distance proportional to the energy of the detected X-ray photons.) At that earlier time I went into a Cambridge electronics and radio-hams’ shop to buy some resistors and there saw a steel frame about 1.5 m 1.5 m 1 m full of 40 cm 40 cm metal-oxide rectifiers, large transformers, and a massive variac (variable transformer) for controlling the output. I was told that this was the filament supply that they were building on a contract for the Mathematical Laboratory for the 3000 valves in the new electronic calculating machine (EDSAC). Hugh Huxley has described how he and John Bennett started to program EDSAC to carry out Fourier syntheses.25 When he left crystallographic work on hemoglobin and moved to his life-long interest in muscle, Kendrew and Bennett took over the development of computer methods.26 However, valve computers were too cumbersome and slow for general laboratory work. It was then generally believed that the future lay with optical and mechanical analogue machines — hence Bernal’s words quoted above. This belief was probably based on the experience that scientists returning from the services had had with mechanical gun-sight predictors and gun controllers. Ray Pepinsky and his team built an electromechanical analogue computer (XRAC) for crystal structure analysis at Pennsylvania State College, whose inauguration in 1951 was celebrated by the conference on ‘‘Computing Methods and the Phase Problem in X-ray Crystal Analysis’’ (Fig. 8). The conference proceedings27 have fascinating details of XRAC itself, which was working for several years, together with papers on other analogue computers such as Beevers’ and Robertson’s Integrator28 (Fig. 9) and on improvements to W. L. Bragg’s29 (1939) optical ‘‘X-ray Microscope.’’ 24
‘‘National Nuclear Energy Series (US).’’ McGraw-Hill, New York, (1946–1950). H. E. Huxley, in ‘‘The Legacy of Sir Lawrence Bragg.’’ (D. C. Phillips and J. M. Thomas, eds.). Science Review London, 1990. 26 J. Bennett and J. C. Kendrew, Acta Crystallogr. 5, 109–116 (1952). 27 R. E. Pepinsky, ‘‘Computing Methods and the Phase Problem in X-ray Crystal Analysis.’’ Dept. of Physics State College, PA, 1952). 28 C. A. Beevers and J. M. Robertson, in ‘‘The Legacy of Sir Lawrence Bragg’’ (D. C. Phillips and J. M. Thomas, eds.), p. 119. Science Review, London, 1990. 29 W. L. Bragg, Nature 143, 678 (1939). 25
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Fig. 8. Participants at R. Pepinsky’s conference on ‘‘Computing Methods,’’ Pennsylvania State College, 1951. Left to right front row: J. M. Bijvoet, Henry Lipson, Caroline McGillavry, Charles Bunn, J. M. Robertson. Back row: Unidentified, Gordon Cox, Max Perutz, Arnold Beevers, Ray Pepinsky, E. Grison. (From Pennsylvania State College.)
There were numerous other analogue devices that have not survived, among them Von Eller’s optical analogue machine30 and Dan MacLachlan Jr.’s31 instrument in which peaks of electron density in a map were built up as sandhills using a stream of sand much as in an hour glass. Buerger’s precession camera32 was a mechanical analogue computer that carried out the transformation of the angular coordinates of a moving crystal into linear coordinates to form a distortion-free projection of the reciprocal lattice. In 1961 David Phillips and I described our linear diffractometer, which transformed linear movements on three orthogonal slides into crystal-shaft rotations (Fig. 5). I remember demonstrating this instrument to P. P. Ewald, explaining to him that it was a mechanical version of 30
G. Von Eller, Compt. Rend. 232, 1122; 233, 2333 (1951). B. Howell, C. J. Christensen, and D. McLachlan, Nature 168, 282 (1951). 32 M. J. Buerger, ‘‘The Precession Method.’’ John Wiley & Sons, New York, 1964. 31
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Fig. 9. Beevers’ and Robertson’s integrator; this device was typical of mechanical analogue computers, some of them derived from wartime gun-sight predictors. (From Pennsylvania State College.)
the Ewald sphere and so his grandchild. Ewald said that he was disappointed to find that he had such an ugly grandchild. We had believed that, as we had no direct access to a digital computer, we needed an analogue device to perform our computations. By the time the commercial version of the linear diffractometer had been launched by Hilger and Watts Ltd, transistor computing circuits had been developed. A little later integrated circuits started to grow exponentially in complexity and the modern digital computer took over. We responded by designing digitally controlled fourcircle X-ray and neutron diffractometers.33 In the first of these automatic four-circle diffractometers (Fig. 10), the crystal shaft was driven by a stepping motor that sounded like a tachycardiac grandfather clock. Sir Lawrence told me that, unknowingly, I had reproduced not only the mechanism but also the sound of his first ionization chamber spectrometer34 (Fig. 11), in which the crystal shaft was connected to a wormwheel that was driven by a shaft fitted with a four-spoke capstan;
33
U. W. Arndt, and B. T. M. Willis, ‘‘Single Crystal Diffractometry.’’ Cambridge University Press, Cambridge, 1966. 34 W. H. Bragg, and W. L. Bragg, Proc. R. Soc. Lond. A88, 428 (1913).
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39
Fig. 10. The author with his first three-circle diffractometer (ca. 1960). (Author’s photograph.)
the operator flicked the capstan in synchronization with a metronome while his eye was glued to the microscope focused on the gold-leaf electroscope that measured the ionization current. By 1964 both the Royal Institution, which I had left in 1963, and the MRC Laboratory of Molecular Biology to which I had moved, had their own computer (each occupying a medium-sized room). We estimate that by 1982 the Laboratory of Molecular Biology had at least four computers and that by 2000 the number was about 600, having probably overtaken the number of scientists and technicians employed in the laboratory. The real trouble with our early computers and with our instruments with paper-tape or punched-card input and output (Fig. 12) was the relative unreliability of these media. At one time I connected three paper-tape punches to my diffractometer in parallel in the hope that suitable editing would produce one usable output tape from our overnight runs. The tapes were handled by assistants known as ‘‘computors’’ who became adept at inserting individual missed holes with a hand-punch and at splicing tapes together.
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Fig. 11. The Bragg ionization spectrometer. The chain was connected to the capstan on the worm-shaft of what would now be called the !-shaft. The microscope was used to view the gold-leaf electrometer connected to the ionization chamber X-ray detector. (Courtesy Royal Institution of Great Britain, the copyright holder.)
The purely electronic circuitry, also, had frequent failures. I remember pointing out to a colleague who was disgusted with the almost weekly breakdowns of one of my diffractometers that its circuitry contained the equivalent of about 1000 transistors, or about 200 times as many as a transistor radio; a mean time between failures of 1 week of the diffractometer
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Fig. 12. Dick Dickerson (right) and Bror Strandberg transporting punched-paper tape computer output from the University Maths Laboratory to the MRC Unit huts. (Courtesy Dr. M. F. Perutz.)
was the equivalent of 4 years’ 24-hr operation of the transistor radio. The advance in automatically controlled instrumentation came only with direct interfaces to small dedicated or embedded computers. The best single-crystal diffractometers still had the disadvantage that they measured only one X-ray reflection at a time. This is not a problem in small-molecule crystallography, where one wants to look at only a relatively small number of widely separated reflections. In large-unit cell diffraction patterns the reflections are close together and many of them occur simultaneously at any given crystal orientation. It is a waste to record these reflections serially one at a time.35 Starting in about 1970 many of us worked on the development of ‘‘area detectors,’’ that is electronic position-sensitive detectors that measured the X-ray intensity at a large number of picture elements (‘‘pixels’’).36 There was considerable rivalry between gas-filled ionization area detectors and those based on television cameras. With the perfection of charge-coupled device (CCD) television cameras, these hold the field at present. 35 36
U. W. Arndt, Acta Crystallogr. B24, 1355 (1968). U. W. Arndt, J. Appl. Crystallogr. 19, 145 (1986).
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Pending the development of efficient electronic area detectors we explored the geometric problems of small-angle rotation patterns by recording them on X-ray film, which was then densitometered on a suitable computer-linked densitometer.37 The technique was successful and commercially produced rotation cameras became generally adopted instruments in their own right for large-molecule crystallography.38 When, later, the storage-phosphor image plate39 replaced silver halide X-ray films, automatic computer-linked image-plate rotation cameras became the instruments of choice. It is only relatively recently that CCD detectors,40 with their advantage of fast read-out, have started to challenge the supremacy of the image plate. Gradually, over the years, computer software for controlling the datacollecting instrumentation and for processing and evaluating the data has replaced the expertise of the crystallographer. Using appropriate programs, protein structures can now often be solved by biologists and biochemists after only a short training in crystallographic methods.41 At one time crystallography was regarded as a separate science in its own right. Today in biology it is seen merely as one of several techniques available for solving structure-related problems. Researchers in the new field of ‘‘structural genomics’’ are looking forward to fully automatic experimentation, from protein isolation and purification via crystallization, crystal harvesting, and mounting to data collection and processing.42 It is all very exciting, but possibly it was more fun in the old days. Acknowledgments I am grateful to the MRC Laboratory where I have spent so many happy years. My particular thanks are due to John Finch and to Michael Fuller for their helpful comments on this chapter. Both are founder members of the Laboratory. Before that, Michael was a member of the ‘‘MRC Unit’’ before it was a ‘‘Laboratory’’ and he has been instrumental in building up its excellent facilities. Their recollections have complemented mine, but the errors and indiscretions in these reflections are purely my own. My thanks also go to those who have kindly given me their permission to reproduce photographs here.
37
U. W. Arndt, J. N. Champness, R. P. Phizackerley, and A. J. Wonacott, J. Appl. Crystallogr. 6, 457 (1973). 38 U. W. Arndt and A. J. Wonacott, eds., ‘‘The Rotation Method in Crystallography.’’ NorthHolland Publishing Co., Amsterdam, 1977. 39 Y. Amemiya and J. Chikawa, Int. Nat. Tab. Crystallogr. 3, Section 7.1.8 I.U.Cr. (1992). 40 S. M. Gruner, Curr. Opin. Struct. Biol. 4, 765 (1994). 41 R. M. Sweet, J. M. Skinner, and M. Cowan, Synthrotron Radiat. News 14(5), 5–11 (2001). 42 Nat. Struct. Genomics Suppl. Nov. 2000.
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˚ . Since then, there have phases were extended all the way from 6 to 3.5 A ˚ been a number of examples where phases have been extended to 3.5 A 24 using merely an initial hollow shell model for a virus to initialize phases ˚ resolution. to about 20 A Conclusion
The reader may have noticed that many of the concepts we now take for granted were initially met with opposition. It is satisfying to see so many of the originally contentious suggestions being now fully accepted. Nevertheless, even now my colleagues and I have had publication difficulties with a paper containing novel methods and unexpected results.25 Acknowledgments A great deal of history is covered in this personal review. It would be impossible to acknowledge all the individuals who have influenced and encouraged me over the past 50 years. I thank all those many individuals who have guided me through problems to a great many successes. Mostly, I have had a lot of fun. Hopefully, I have been able to help others to enjoy their scientific endeavors. One of the greatest rewards has been to have found lasting friendships in almost every part of the world. I thank primarily the NIH, NSF, and Purdue University for continuing financial support.
24
J. Tsao, M. S. Chapman, M. Agbandje, W. Keller, K. Smith, H. Wu, M. Luo, T. J. Smith, M. G. Rossmann, R. W. Compans, and C. R. Parrish, Science 251, 1456 (1991). 25 R. J. Kuhn, W. Zhang, M. G. Rossmann, S. V. Pletnev, J. Corver, E. Lenches, C. T. Jones, S. Mukhopadhyay, P. R. Chipman, E. G. Strauss, T. S. Baker, and J. H. Strauss, Cell 105, 127 (2001).
[3] Personal X-ray Reflections By U. W. Arndt One of the results of having been around for quite a long time and of having met many of the great figures of the past is that one gets asked to write historical surveys of one’s subject. To do so properly is not at all easy: according to Horace Judson, author of the majestic history of Molecular Biology, ‘‘The Eighth Day of Creation,’’ practicing scientists are poor witnesses. They like to look back on their own work as though it had all conformed to a well-thought out plan in which each advance followed from the previous one, itself, of course, preferably made by themselves or their pupils.
METHODS IN ENZYMOLOGY, VOL. 368
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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In practice, of course, progress has often depended on luck and on the timeliness of the project, that is, on the availability of the necessary tools and techniques, and on the political atmosphere that encourages funding of the field. Accordingly, I shall not attempt to write a serious historical survey along the lines of that by Brian Matthews1 in an earlier volume of this series. I shall, instead, merely try to record my reflections on some of the events I was fortunate enough to witness or be involved in myself. Much of my working life has been spent in Cambridge and so my account is naturally biased toward what happened there. I began working in crystallography (but not in protein crystallography) in 1944 in the Cavendish Laboratory Cambridge, where Max Perutz had already started on his self-imposed task of determining the crystal structure of hemoglobin. At that time many small-molecular crystallographers thought that this was an insoluble problem: Even if by some miracle he managed to measure the intensity of tens of thousands of X-ray reflections, and even if by some further miracle the phases of those reflections were revealed to him, it would take 20 years of churning away on the mechanical calculating machines of the day to calculate the electron density in order to plot three-dimensional maps of the molecule. Besides, there was no guarantee that the molecule, which, after all, reacted only when in solution, would have the same shape when in that state as when in a solid crystal. I once asked Max Perutz whether at that time he had an inkling that computers were just around the corner. He told me, no, he just carried on on his chosen path, hopeful that each problem would be solved in turn. The world acknowledges a project as having been successful only when it is conducted to a conclusion. In his Nobel Prize lecture in 19622 Perutz quoted Sir Francis Drake’s famous prayer . . . . ‘‘Grant us also to know that it is not the beginning but the continuing [of any great matter] until it is thoroughly finished which yieldeth the true glory.’’ This is an excellent maxim except in those cases when one is hitting one’s head against a brick wall. The mark of successful researchers is that they recognize the brick wall for what it is and that they then switch to a more promising problem.3 Mere ideas are ten-a-penny, as Ken Holmes was fond of saying. This is particularly worth remembering today when what might be a solution is arrived at by guess-work, now called computer simulation, and when the real answer must be checked by practical experiment. I am reminded of an example in electron microscopy. 1
B. W. Matthews, Methods Enzymol. 276, 3 (1997). M. F. Perutz, Nobel Prize Lecture, Stockholm (1962). 3 A. D. McLachlan, Personal Communication. 2
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At a conference on electron microscopy in 19704 Walter Hoppe of Munich presented a minutely argued treatise on the benefits in resolution and contrast which could be expected by placing a mask in the direct electron beam, provided this opaque mask was exactly centered. The next speaker was Nigel Unwin who showed actual examples of the improvements which he had achieved by the much simpler procedure of mounting a gold-coated spider’s thread at the exit of the objective lens. ‘‘But how did you succeed in exactly centering the thread?’’ Hoppe wanted to know. The answer, ‘‘Oh, we just made the spider walk across the hole,’’ led to a collapse of the audience in laughter. Poor Walter Hoppe! He was a very nice man as well as being a brilliant scientist who made important contributions in many fields ranging from instrument development and X-ray optics to phase determination, but he tended to be caught on the wrong foot at conferences. There was the occasion when he commented on a talk by Caroline MacGillavry, whose name he mispronounced Macgilla´ vry several times. She did not correct him, but in her reply she referred to him only as Professor Hop. But let us return to protein crystallography. I was fortunate in visiting Cambridge from London where I was then working at the Royal Institution, a day or two after Perutz and his team had obtained precession photographs of a crystal of native horse haemoglobin and of an isomorphous crystal of a derivative in which two mercury atoms had been attached to the protein molecule.5 Laying the two photographs over one another with a slight displacement it was immediately obvious that there were intensity differences: in principle, the phase problem had been solved, and the path, though still a long one, lay open for a complete structure determination. Everyone in what was then the MRC Unit was in a state of euphoria and Max Perutz seemed to be floating inches above the floor. X-ray tubes in the early days were not very powerful and exposure times of several days were sometimes necessary. The maximum power that can be dissipated in the tube target without melting it is set by the rate at which heat can be conducted away from the electron focus to the watercooled rear surface of the target. A higher power is possible when the target surface is moved. Starting with those of A. Mu¨ ller,6 several designs of rotating-anode tubes were produced, including Hoppe’s highly sophisticated idea for an anode cooled with liquid gallium or sodium, which has never been developed further. However, although these generators allowed a higher power dissipation in the target, the brilliance of the source 4
Reported in Phil. Trans. R. Soc. Lond. B, 261 (1971). D. W. Green, V. Ingram, and M. F. Perutz, Proc. R. Soc. Lond. A225, 287 (1954). 6 A. Mu¨ ller, Proc. R. Soc. Lond. A125, 507 (1929). 5
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was mostly low, that is, the electron focus on the target was too large to give much improvement in the intensity that reached a small crystal. The 50-kW continuously-pumped rotating-anode tube built by Mu¨ ller at the Royal Institution (Fig. 1) had a foreshortened electron focus several millimeters in diameter; it actually delivered a lower X-ray intensity to a small sample than a 1-kW sealed-off X-ray tube. One of my first acts on starting work at the Royal Institution in 1950 was to recommend the retirement of the monster. We were then able to work in the laboratory without earmuffs to reduce the roar from the 10 horsepower motor and the screech from the stuffing box vacuum seal for the target shaft. Incidentally, the laboratory in which the tube was housed was used by Michael Faraday and in 1950 it still contained some of his apparatus, which looked as though it had not been touched since his days. In the cold winters in the late 1940s Lonsdale and Owston carried on their work in this room: their publication7 contains the statement that the work was carried out at room temperature ( 5 C). Today the room has been restored as the Faraday Museum.
Fig. 1. Mu¨ ller’s rotating-anode X-ray generator at the Royal Institution, London, ca. 1950. The 10-horsepower motor on the right of the bed drove the 600-mm-diameter target via a long shaft running in a vaccum-tight stuffing box. The hexaphase power supply, with water-cooled rectifier valves, was in the cubicle on the left. Vacuum pumps for the tube were under the bench. (Author’s photograph.)
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A compact rotating-anode tube was designed by A. Taylor8 and was further improved by Tony Broad in the MRC Unit in Cambridge.9 Finally, Hugh Huxley and Ken Holmes combined Broad’s anode with a better electron gun based on that of the French Beaudouin tube. This hybrid tube was produced commercially by Elliot Brothers in England, later by GEC, and finally by Enraf Nonius, and was for many years the standard workhorse for protein crystallography. Recently, high-brilliance microsource X-ray tubes have been reintroduced.10 It was well known that a very small electron focus allowed more efficient cooling of the X-ray tube target as the isothermal surfaces in this target became hemispherical rather than planar. Advances in X-ray optics allowed the radiation to be focused on the specimen crystal.11 With suitable X-ray mirrors the focused intensity with a low-power microfocus tube can be made to exceed the intensity in the aperture-limited unfocused beam from a rotating-anode X-ray generator operated at more than 100 times the power. Focusing ellipsoidal and paraboloidal X-ray mirrors had been produced in Prague for X-ray telescopes flown in Russian satellites. My request for reprints brought the literature, accompanied by a puzzled inquiry as to why someone in a laboratory of molecular biology was interested in X-ray astronomy. In due course this led to a fruitful collaboration that resulted in the production of a series of mirrors for use with microfocus tubes.12 It is difficult today to remember the manipulatory skills that were required to mount and align protein crystals on the much cruder X-ray goniometer heads and on the X-ray cameras that lacked some of the modern facilities (Fig. 2), and there was much to learn about the behavior of protein crystals. It seems highly appropriate that it was Francis Crick, who was much given to handwaving (Fig. 3), who discovered that draughts and temperature gradients near a mounted crystal would lead to a distillation of solvent from one part of the specimen tube to another and thus to changes of unit cell parameters.13
7
K. Lonsdale and P. Owston, J. Glaciol. (1948). A. Taylor, J. Sci. Instrum. 26, 225 (1949); Rev. Sci. Instrum. 27, 757 (1956). 9 D. A. G. Broad, Rotating-anode X-ray tube. UK Patent Applications, Nos. 5172, 5173, 12761, 38939, 13376 (1956). 10 U. W. Arndt, J. V. P. Long, and P. Duncomb, J. Appl. Crystallogr. 31, 936 (1998). 11 U. W. Arndt, P. Duncomb, J. V. P. Long, L. Pina, and A. Inneman, J. Appl. Crystallogr. 31, 733 (1998). 12 L. Pina, A. Inneman, R. Hudec, U. W. Arndt, N. Loxley, G. Fraser, M. Taylor, and J. Wall, Proc. SPIE 4144, 165 (2000). 13 F. H. C. Crick and B. S. Magdoff, Acta Crystallogr. 9, 901 (1956). 8
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Fig. 2. Unicam single-crystal X-ray camera in the foreground, with cylindrical film holder beside it. Until about 1955 most protein photographs were taken with this camera (in the background a Philips Debye-Scherrer camera is seen mounted at another window of a vertically mounted sealed-off X-ray tube). (From LMB Archives.)
The real value of protein crystallography started to emerge only when the first structures were carried as far as atomic resolution. Only then did it become apparent that the molecule as seen by the crystallographer had the shape that it took up in solution and that a knowledge of the shape of an enzyme molecule could explain the nature of enzyme–substrate interactions. Lower-resolution maps on which individual atoms could not be identified still contained many ambiguities. Before it was realized that proteins consisted of long folded polypeptide chains the ‘‘cyclol’’ theory of protein structure, which postulated interlocking rings or cages, had some ˚ resolution map of myoglobin had come adherents. By the time the 5-A out the cyclol theory was largely discredited, but I once witnessed Isidore Fankuchen standing in front of this map and, with his tongue in his cheek, pointing out how clearly visible the cyclols were! In the 1960s some people believed that the successes of X-ray crystal structure determination were so spectacular that it would soon become the standard technique for amino acid sequence analysis. This has not
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Fig. 3. Francis Crick, waving his hands (see text). (From LMB Archives and Prof. Uno Lindberg.)
happened, partly, of course, because sequencing techniques have also been greatly improved and automated since Fred Sanger’s heroic efforts in sequencing insulin, for which he was awarded his first Nobel Prize. At the party following his second Nobel Prize in 1980 for base sequencing in DNA several speakers spoke of Fred’s unassuming modesty. After a good deal of champagne had flowed, Fred was forced to reply to his well-wishers, which he did with the following words: ‘‘Everyone says how modest I am; I may be modest, but I am also bloody good.’’ The applause nearly brought the roof down. The solution of the structures of myoglobin by Kendrew and of hemoglobin by Perutz (Fig. 4), followed in 1965 by that of the first enzyme, lysozyme, by David Phillips and his team at the Royal Institution,14 was a great stimulus to look at the structure of other proteins. Sir Lawrence Bragg, the Cavendish professor, had been giving enthusiastic moral support to the hemoglobin group, which became the MRC Unit for the Study of Molecular Structure of Biological Systems in 1947 and, in 1962, the MRC Laboratory of Molecular Biology (LMB). Based on his own earlier pioneer work 14
C. C. F. Blake, D. F. Koenig, G. A. Mair, A. C. T. North, D. C. Phillips, and V. R. Sarma, Nature 206, 757 (1965).
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Fig. 4. Max Perutz (left) and John Kendrew, sketched by W. L. Bragg, ca. 1955. (From LMB Archives.)
on the families of silicate minerals,15 Bragg believed that the elucidation of a few protein structures would reveal the common features of all proteins and thus ‘‘explain the Secret of Life.’’ In this belief he was wrong: different proteins have turned out to be as varied and as fiercely individualistic as their investigators. I should like to express a heretical belief of my own here. Certainly Bragg was interested in the Secret of Life, but I believe that his primary interest in protein structure was different: his career had been a splendid progress from the simplest crystal structure of all, that of sodium chloride, to the structures of metals and minerals. He was now faced with the largest and most complex molecules known to man, and that is what excited him. Most of the early protein crystallographers had started life as physicists or physical chemists and were essentially interested in the structures as structures, and not then as stepping stones to the answering of biological questions. It needed the entry into the field of biochemists, geneticists, and other biologists to ask these questions and to find answers to them. It would be nice to think that all the new instruments and techniques that were developed by physicists and engineers who moved into molecular biology were part of a logical program devised after experimental bottlenecks had been identified. In fact, I suspect that many instrument makers like myself were partly motivated by a desire to continue to play with the Meccano construction sets and model railways of our childhood. It was sometimes difficult to know when to stop perfecting an instrument and to 15
W. L. Bragg, ‘‘The Atomic Structure of Minerals.’’ Cornell University Press, New York, (1937).
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start using it for actual research. When David Phillips and I had built our linear diffractometer16 (Fig. 5), I would have gone on making small improvements when David started to collect data from a myoglobin crystal, in accordance with his oft-repeated saying ‘‘The best is the enemy of the good.’’ The ‘‘Secret of Life’’ made a comeback many years later when the protagonists of new synchrotron radiation sources were looking for scientific allies among biologists. The synchrotron beams were sufficiently intense to offer a promise of investigating structure changes in biological materials dynamically, and, after all, it was the undergoing of changes that distinguished life and biological materials. A questioning of what interesting biological systems there were in which changes should be investigated usually provided the answer: ‘‘well, there is muscle and er . . . er . . . muscle . . . and muscle . . . !’’ Whatever the truth is in these speculations, the search was on for interesting proteins to investigate. The path soon led away from the farmyard: John Kendrew had an early arrangement with the London Zoo that they would notify him of the deaths of unusual diving animals such as penguins, seals, and sea-lions, from whose carcasses he would extract myoglobin. David Blow obtained his cytochrome c samples from tuna fish hearts, but prizes awarded by colleagues would probably have gone to Herman Watson who looked at lobster GPD (glyceraldehyde phosphodehydrogenase). A consignment of fresh West Country lobsters arrived at intervals for processing and for extracting the protein. Fortunately, for a reason not clear to me, the claws were not suitable for this purpose and were thus available for distribution to Herman’s friends. Not all attempts to obtain proteins from raw materials were equally successful. Francis Crick at one time organized a group of us to sit in a circle and breathe in the fumes from freshly cut onions. He collected our tears, but when he came to analyze them they contained very little lysozyme as hoped, but were almost pure onion juice. The most attractive raw materials are not always the most suitable ones for scientific purposes. In 1954 Dennis Riley gave a Royal Institution Friday Evening Discourse17 on the subject ‘‘The Raw Materials of Life.’’18 Several of the lecture demonstrations and experiments involved hen eggs, at that time still rationed in Britain. However, the Egg Marketing Board was sympathetic and assisted Riley in obtaining a supply of particularly large and beautiful eggs for such a high scientific purpose. These 16
U. W. Arndt and D. C. Phillips, Acta Crystallogr. 14, 807 (1961). D. P. Riley, Proc. R. Inst. Gt. Brit. 35, 363 (1952). 18 D. P. Riley and U. W. Arndt, Nature 171, 144 (1953). 17
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Fig. 5. The first manual linear diffractometer, ‘‘P. P. Ewald’s grandchild.’’ (Author’s photograph.)
‘‘Discourses’’ were important social occasions and it was pretty daunting for the lecturer to enter the ‘‘Amphitheatre’’ at 9 o’clock sharp to be faced with rows upon rows of the distinguished audience in full evening dress. The lecturer frequently restored his courage with a stiff double whisky just before his ordeal. Riley’s first demonstration was to break an egg into a bowl of oil to show the separation into yolk and eggwhite. The extra-large egg turned out to have a double yolk, as by a rare chance did the next one. The lecturer was clearly extremely concerned whether he was seeing things and his enormous relief was obvious when he caught murmurs from the audience ‘‘look, another double-yolked egg.’’
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I can remember another time when the source of the material could have been, but was not, of great importance. In 1971 Ken Holmes with his co-workers Rosenbaum and Witz from Heidelberg had built a beamline to exploit the synchrotron radiation from a port of the new German electron synchrotron (DESY) in Hamburg and I had gone there to assist them in getting what was to be the first synchrotron radiation diffraction photograph of a muscle specimen.19 We worked all day to get everything ready for the moment when the beam was switched on for the evening shift. Holmes had a gentleman’s agreement with his former colleague Hugh Huxley, who had developed the mirrors and monochromators and the techniques for examining muscles (Fig. 6), that frog sartorius muscle belonged to Hugh; Ken would concentrate on insect flight muscle. (Such gentleman’s agreements were common in the early days, when it was not ‘‘done’’ to start a structure determination of a protein on which someone else was known to be working.) Accordingly, Holmes and his team brought along a specimen of a waterbeetle flight muscle, which had not been photographed properly before at a conventional X-ray source. With some effort Ken was persuaded that it would be particularly valuable to take one diffraction photograph of a well-known material in order to make quantitative comparisons of the two sources. As evening fell, Gerd Rosenbaum was despatched to go frog-hunting at a neighboring pond in company with the DESY gatekeeper’s son. They failed to catch a frog, but fortunately the insect flight muscle photograph turned out to be a sufficiently good one to make a powerful case for the continued development of synchrotron sources at DESY and elsewhere. Holmes’ synchrotron radiation work had a political as well as a scientific importance. John Kendrew had, for some time, been arguing the case for a European Molecular Biology Laboratory (EMBL), which, like CERN (Centre Europe´ en pour la Recherche Nucle´ aire), would be able to undertake research too expensive for individual national laboratories. He came up against opposing claims that there was really nothing sufficiently expensive in molecular biology to justify an international laboratory. The highcost synchrotron radiation work came at the right moment and EMBL was planned at the outset with a main laboratory at Heidelberg and an outstation at DESY. There was and is another EMBL outstation at Grenoble, France, adjacent to the Institut Laue-Langevin (ILL), to benefit from collaborative biological structure work with neutrons from the high-flux beam reactor at the latter institute. The existence of the administrative machinery at the trinational (French, German, and British) ILL was one of the reasons adduced for building the European Synchrotron Radiation Facility 19
G. Rosenbaum, K. C. Holmes, and J. Witz, Nature 230, 129 (1971).
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Fig. 6. H. E. Huxley, adjusting his muscle camera. (From LMB Archives.)
(ESRF) next door to the ILL instead of in Strasbourg, which would have been the site preferred by many French scientists. The decision led to considerable but unsuccessful agitation in Alsace with car stickers and posters protesting against the ‘‘betrayal’’ of Alsace and its capital Strasbourg in depriving it of the synchrotron. Following the work of Holmes at DESY and of Keith Hodgson and his colleagues at Stanford,20 protein crystallographers were quick to realize the advantages of the X-ray flux, which was many times greater than that from conventional laboratory X-ray sources. Before long they started to 20
J. C. Phillips, A. Wlodawar, M. M. Yewitz, and K. O. Hodgson, Proc. Nat. Acad. Sci. USA 73, 128 (1976).
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appreciate the benefits of the tuneability of synchrotron beam lines, which ˚ X-rays. meant that they were no longer exclusively limited to 1.54-A In due course the success of synchrotron radiation sources in the United States, Germany, the UK, Japan, and France led to the demand for more and more ‘‘protein crystallography (PX)’’ beam lines and thus, to a considerable extent, to the plan for a European Synchrotron Research Facility ‘‘to keep up with the Americans’’ and for a National Synchrotron Light Source (NSLS) and an Advanced Photon Source (APS) in the United States ‘‘to keep up with the Europeans.’’ The growth of synchrotron radiation research is illustrated by the fact that the (2001) Editorial Board of Synchrotron Radiation News has members in synchrotron radiation laboratories in 15 countries, all of which have crystallographers who use synchrotron radiation. Many protein crystallographers now think little of making several multithousand mile trips a year to collect diffraction data at a center where they can find a suitable beam line. More importantly, funding agencies are prepared to finance such trips. Data collection runs are now sometimes carried out in as many hours as the number of weeks necessary in earlier days. The development of these shared resources accelerated the internationalization of what had always been a field in which one’s collaborators came from many different countries. One of the privileges of having worked in the field of molecular biology is that there is virtually no country in the six continents in which one cannot find close colleagues and good friends. With the projected zero-gravity protein crystallization and protein structure determination program in the International Space Laboratory such colleagues may soon not be restricted to the earth’s surface. Protein crystallography had first taken off in Britain, where for a surprisingly long time nearly all crystallographers could trace their scientific descent from either W. H. or W. L. Bragg. The explosive growth of the subject in the early and mid-1950s owed much to overseas visitors and postdoctoral workers, especially those from the United States. At the celebration of the twenty-fifth anniversary of the Nobel Prizes to Watson, Crick, Perutz, and Kendrew, which was also the twenty-fifth birthday of the MRC Laboratory of Molecular Biology in Cambridge, and the fortieth birthday of the original MRC Unit, Jim Watson noted that a high proportion of major American professorships of Molecular Biology were filled by alumni of the laboratory. Today news is sent to overseas collaborators by e-mail so quickly that it is often not read before being replied to. In the early days news traveled almost as fast by word of mouth. In 1976 a committee set up in Britain to investigate the efficiency of government-financed laboratories noted that
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the MRC Laboratory of Molecular Biology had an unusually small bill for textbooks and journals. The explanation that ‘‘when someone elsewhere had done something interesting he usually came to Cambridge to tell us about it’’ was received with skepticism. However, this was one of the facts that made for particularly lively seminars with hard-hitting discussions. Sydney Brenner’s not-so sotto voce comments often enlivened a colloquium; Francis Crick, just back from a visit to Leslie Orgel at La Jolla, once reported on Leslie’s latest ideas there that life had started in the primeval soup as chips of apatite and was interrupted by the remark ‘‘I see, chips before fish.’’ Chips also had another influence on molecular biology. Protein crystallography was transformed by the advent of electronic computing; it was arguably one of the first scientific fields that made constant use of computers. Of course, computers could be developed only when the whole field of electronics had advanced sufficiently and, in particular, they needed the introduction of transistors and integrated solid-state circuitry. The development is still far from complete: Ge´ rard Bricogne once described human beings as ‘‘the catalyst in the transition from carbon-based to silicon-based intelligence.’’21 It is difficult to appreciate just how great the developments in electronics and in computing have been. Strangely, while our laboratories had excellent mechanical workshops and instrument makers, it was not for many years that adequate space and manpower were devoted to electronic design and construction. In 1944 there was not a single ‘‘valve’’ (electronic vacuum tube) in the crystallographic laboratory of the Cavendish Laboratory, Cambridge. Fourier syntheses were carried out with the help of Beevers–Lipson strips22 (already a great advance on having to look up sines and cosines in books of tables), and only the senior research students had easy access to motor-driven adding machines. The rest of us had to content ourselves with the slower manual adding machines. The motor-driven calculators had another advantage, discovered by Alex Stokes: when the darkroom clock broke down Alex worked out an appropriate long division so that the silence at the end of its labors signaled the end of the development period. In 1945 J. D. Bernal spoke at a British Institute of Physics Conference on the ‘‘The Future of X-ray Analysis.’’23 He said ‘‘there must be a great development of analytic methods and means of calculation. We are only at the beginning of mechanical and optical methods, and their 21
G. Bricogne, Personal communication. C. A. Beevers and H. Lipson, Proc. Phys. Soc. Lond. 48, 772 (1936). 23 J. D. Bernal, Nature 155, 713 (1945). 22
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improvement is very necessary as calculations are becoming a bottleneck. There is no use in being able to take (X-ray) photographs in a few seconds if calculations take months.’’ The progress in electronic computers depended on the progress of electronics as such (Fig. 7). In about 1945 when I started to develop my own X-ray Geiger counters I had, of course, to build my own electronic amplifiers and counters. Manhattan project electronic designs24 had not yet been declassified, and ‘‘counters’’ were called ‘‘scalers’’ because they
Fig. 7. Construction of XRAC. Electronic instruments were usually assembled from interconnected 19-in. chasses like the ones illustrated here. Today such a chassis, filled with dozens of tubes, could be replaced by one integrated circuit. (From Pennsylvania State College.)
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scaled down the counting rate by 32 or 64 to a rate with which Post Office telephone message registers could cope. (Some years later I started playing with energy-sensitive proportional counters and needed a pulseheight-analyzer; this was known as a ‘‘Kick Sorter’’ because such a device had been built at the Cavendish in which the pulses from the radiation detector were amplified and fed into a loud-speaker coil where they served to catapult —‘‘kick’’ — small steel balls a distance proportional to the energy of the detected X-ray photons.) At that earlier time I went into a Cambridge electronics and radio-hams’ shop to buy some resistors and there saw a steel frame about 1.5 m 1.5 m 1 m full of 40 cm 40 cm metal-oxide rectifiers, large transformers, and a massive variac (variable transformer) for controlling the output. I was told that this was the filament supply that they were building on a contract for the Mathematical Laboratory for the 3000 valves in the new electronic calculating machine (EDSAC). Hugh Huxley has described how he and John Bennett started to program EDSAC to carry out Fourier syntheses.25 When he left crystallographic work on hemoglobin and moved to his life-long interest in muscle, Kendrew and Bennett took over the development of computer methods.26 However, valve computers were too cumbersome and slow for general laboratory work. It was then generally believed that the future lay with optical and mechanical analogue machines — hence Bernal’s words quoted above. This belief was probably based on the experience that scientists returning from the services had had with mechanical gun-sight predictors and gun controllers. Ray Pepinsky and his team built an electromechanical analogue computer (XRAC) for crystal structure analysis at Pennsylvania State College, whose inauguration in 1951 was celebrated by the conference on ‘‘Computing Methods and the Phase Problem in X-ray Crystal Analysis’’ (Fig. 8). The conference proceedings27 have fascinating details of XRAC itself, which was working for several years, together with papers on other analogue computers such as Beevers’ and Robertson’s Integrator28 (Fig. 9) and on improvements to W. L. Bragg’s29 (1939) optical ‘‘X-ray Microscope.’’ 24
‘‘National Nuclear Energy Series (US).’’ McGraw-Hill, New York, (1946–1950). H. E. Huxley, in ‘‘The Legacy of Sir Lawrence Bragg.’’ (D. C. Phillips and J. M. Thomas, eds.). Science Review London, 1990. 26 J. Bennett and J. C. Kendrew, Acta Crystallogr. 5, 109–116 (1952). 27 R. E. Pepinsky, ‘‘Computing Methods and the Phase Problem in X-ray Crystal Analysis.’’ Dept. of Physics State College, PA, 1952). 28 C. A. Beevers and J. M. Robertson, in ‘‘The Legacy of Sir Lawrence Bragg’’ (D. C. Phillips and J. M. Thomas, eds.), p. 119. Science Review, London, 1990. 29 W. L. Bragg, Nature 143, 678 (1939). 25
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Fig. 8. Participants at R. Pepinsky’s conference on ‘‘Computing Methods,’’ Pennsylvania State College, 1951. Left to right front row: J. M. Bijvoet, Henry Lipson, Caroline McGillavry, Charles Bunn, J. M. Robertson. Back row: Unidentified, Gordon Cox, Max Perutz, Arnold Beevers, Ray Pepinsky, E. Grison. (From Pennsylvania State College.)
There were numerous other analogue devices that have not survived, among them Von Eller’s optical analogue machine30 and Dan MacLachlan Jr.’s31 instrument in which peaks of electron density in a map were built up as sandhills using a stream of sand much as in an hour glass. Buerger’s precession camera32 was a mechanical analogue computer that carried out the transformation of the angular coordinates of a moving crystal into linear coordinates to form a distortion-free projection of the reciprocal lattice. In 1961 David Phillips and I described our linear diffractometer, which transformed linear movements on three orthogonal slides into crystal-shaft rotations (Fig. 5). I remember demonstrating this instrument to P. P. Ewald, explaining to him that it was a mechanical version of 30
G. Von Eller, Compt. Rend. 232, 1122; 233, 2333 (1951). B. Howell, C. J. Christensen, and D. McLachlan, Nature 168, 282 (1951). 32 M. J. Buerger, ‘‘The Precession Method.’’ John Wiley & Sons, New York, 1964. 31
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Fig. 9. Beevers’ and Robertson’s integrator; this device was typical of mechanical analogue computers, some of them derived from wartime gun-sight predictors. (From Pennsylvania State College.)
the Ewald sphere and so his grandchild. Ewald said that he was disappointed to find that he had such an ugly grandchild. We had believed that, as we had no direct access to a digital computer, we needed an analogue device to perform our computations. By the time the commercial version of the linear diffractometer had been launched by Hilger and Watts Ltd, transistor computing circuits had been developed. A little later integrated circuits started to grow exponentially in complexity and the modern digital computer took over. We responded by designing digitally controlled fourcircle X-ray and neutron diffractometers.33 In the first of these automatic four-circle diffractometers (Fig. 10), the crystal shaft was driven by a stepping motor that sounded like a tachycardiac grandfather clock. Sir Lawrence told me that, unknowingly, I had reproduced not only the mechanism but also the sound of his first ionization chamber spectrometer34 (Fig. 11), in which the crystal shaft was connected to a wormwheel that was driven by a shaft fitted with a four-spoke capstan;
33
U. W. Arndt, and B. T. M. Willis, ‘‘Single Crystal Diffractometry.’’ Cambridge University Press, Cambridge, 1966. 34 W. H. Bragg, and W. L. Bragg, Proc. R. Soc. Lond. A88, 428 (1913).
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Fig. 10. The author with his first three-circle diffractometer (ca. 1960). (Author’s photograph.)
the operator flicked the capstan in synchronization with a metronome while his eye was glued to the microscope focused on the gold-leaf electroscope that measured the ionization current. By 1964 both the Royal Institution, which I had left in 1963, and the MRC Laboratory of Molecular Biology to which I had moved, had their own computer (each occupying a medium-sized room). We estimate that by 1982 the Laboratory of Molecular Biology had at least four computers and that by 2000 the number was about 600, having probably overtaken the number of scientists and technicians employed in the laboratory. The real trouble with our early computers and with our instruments with paper-tape or punched-card input and output (Fig. 12) was the relative unreliability of these media. At one time I connected three paper-tape punches to my diffractometer in parallel in the hope that suitable editing would produce one usable output tape from our overnight runs. The tapes were handled by assistants known as ‘‘computors’’ who became adept at inserting individual missed holes with a hand-punch and at splicing tapes together.
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Fig. 11. The Bragg ionization spectrometer. The chain was connected to the capstan on the worm-shaft of what would now be called the !-shaft. The microscope was used to view the gold-leaf electrometer connected to the ionization chamber X-ray detector. (Courtesy Royal Institution of Great Britain, the copyright holder.)
The purely electronic circuitry, also, had frequent failures. I remember pointing out to a colleague who was disgusted with the almost weekly breakdowns of one of my diffractometers that its circuitry contained the equivalent of about 1000 transistors, or about 200 times as many as a transistor radio; a mean time between failures of 1 week of the diffractometer
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Fig. 12. Dick Dickerson (right) and Bror Strandberg transporting punched-paper tape computer output from the University Maths Laboratory to the MRC Unit huts. (Courtesy Dr. M. F. Perutz.)
was the equivalent of 4 years’ 24-hr operation of the transistor radio. The advance in automatically controlled instrumentation came only with direct interfaces to small dedicated or embedded computers. The best single-crystal diffractometers still had the disadvantage that they measured only one X-ray reflection at a time. This is not a problem in small-molecule crystallography, where one wants to look at only a relatively small number of widely separated reflections. In large-unit cell diffraction patterns the reflections are close together and many of them occur simultaneously at any given crystal orientation. It is a waste to record these reflections serially one at a time.35 Starting in about 1970 many of us worked on the development of ‘‘area detectors,’’ that is electronic position-sensitive detectors that measured the X-ray intensity at a large number of picture elements (‘‘pixels’’).36 There was considerable rivalry between gas-filled ionization area detectors and those based on television cameras. With the perfection of charge-coupled device (CCD) television cameras, these hold the field at present. 35 36
U. W. Arndt, Acta Crystallogr. B24, 1355 (1968). U. W. Arndt, J. Appl. Crystallogr. 19, 145 (1986).
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Pending the development of efficient electronic area detectors we explored the geometric problems of small-angle rotation patterns by recording them on X-ray film, which was then densitometered on a suitable computer-linked densitometer.37 The technique was successful and commercially produced rotation cameras became generally adopted instruments in their own right for large-molecule crystallography.38 When, later, the storage-phosphor image plate39 replaced silver halide X-ray films, automatic computer-linked image-plate rotation cameras became the instruments of choice. It is only relatively recently that CCD detectors,40 with their advantage of fast read-out, have started to challenge the supremacy of the image plate. Gradually, over the years, computer software for controlling the datacollecting instrumentation and for processing and evaluating the data has replaced the expertise of the crystallographer. Using appropriate programs, protein structures can now often be solved by biologists and biochemists after only a short training in crystallographic methods.41 At one time crystallography was regarded as a separate science in its own right. Today in biology it is seen merely as one of several techniques available for solving structure-related problems. Researchers in the new field of ‘‘structural genomics’’ are looking forward to fully automatic experimentation, from protein isolation and purification via crystallization, crystal harvesting, and mounting to data collection and processing.42 It is all very exciting, but possibly it was more fun in the old days. Acknowledgments I am grateful to the MRC Laboratory where I have spent so many happy years. My particular thanks are due to John Finch and to Michael Fuller for their helpful comments on this chapter. Both are founder members of the Laboratory. Before that, Michael was a member of the ‘‘MRC Unit’’ before it was a ‘‘Laboratory’’ and he has been instrumental in building up its excellent facilities. Their recollections have complemented mine, but the errors and indiscretions in these reflections are purely my own. My thanks also go to those who have kindly given me their permission to reproduce photographs here.
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U. W. Arndt, J. N. Champness, R. P. Phizackerley, and A. J. Wonacott, J. Appl. Crystallogr. 6, 457 (1973). 38 U. W. Arndt and A. J. Wonacott, eds., ‘‘The Rotation Method in Crystallography.’’ NorthHolland Publishing Co., Amsterdam, 1977. 39 Y. Amemiya and J. Chikawa, Int. Nat. Tab. Crystallogr. 3, Section 7.1.8 I.U.Cr. (1992). 40 S. M. Gruner, Curr. Opin. Struct. Biol. 4, 765 (1994). 41 R. M. Sweet, J. M. Skinner, and M. Cowan, Synthrotron Radiat. News 14(5), 5–11 (2001). 42 Nat. Struct. Genomics Suppl. Nov. 2000.