Crystallogenesis Research for Biology in the Last Two Decades as Seen from the International Conferences on the Crystallization of Biological Macromolecules† Alexander McPherson‡ and Richard Giegé*,#
CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 11 2126–2133
Department of Molecular Biology and Biochemistry, UniVersity of California IrVine, IrVine, California 92697-3900, and UPR 9002, IBMC du CNRS & UniVersité Louis Pasteur, 15, rue René Descartes, F-67084 Strasbourg, France ReceiVed July 23, 2007; ReVised Manuscript ReceiVed September 4, 2007
ABSTRACT: The series of 11 International Conferences on the Crystallization of Biological Macromolecules (ICCBM) took place over the period 1986–2006 in the USA (four times), Germany (two times), China, France, Japan, Spain, and lastly the 11th in Canada in Quebec City. Here we review the first 10 ICCBMs. Their focus was to bring rational approaches to the field of protein crystal growth and thus overcome the rate-limiting step in macromolecular X-ray crystallography. This survey summarizes how the ICCBM series contributed to the emergence of the science of biocrystallogenesis. This was achieved through the joint efforts of scientists from the small molecule crystal growth community and from biochemists, biophysicists, and protein crystallographers. Highlights from each conference are discussed, and scientific synergies are emphasized. While the first conferences focused on fundamentals, especially from the standpoint of physics and biochemical considerations, the more recent conferences stressed applications in structural biology, to advanced methods of crystallization, and of crystal quality improvement. Particular attention will be given to themes that were recurrent through all the ICCBMs: purity and impurities, solution properties of macromolecules under precrystallization conditions, microgravity and assessment of crystal quality, as well as specific trends of practical interest to structural biology.
1. Introduction The name crystallogenesis arose when it became evident that the field of crystallization of proteins and other biological macromolecules was not restricted simply to crystal production for diffraction studies, but it encompassed, in fact, all phases of structural biology, from protein expression and purification, to recording of diffraction data. The term was initially suggested at the very first conference,1 where it challenged the ears of crystal growth physicists but had an agreeable ring to biologists. Currently, it is widely accepted by the community. Indeed, crystallogenesis, as reflected by the International Conference on the Crystallization of Biological Macromolecules (ICCBM) series, developed progressively into an interdisciplinary area in science and engineering between crystallography, biochemistry, physics and biophysics, materials, crystal growth, colloidal and polymer science, and informatics as was summarized by Alex Chernov and Larry DeLucas at ICCBM-9 in Jena.2 Presently, molecular biology and macromolecular engineering, as well as the miniaturization of crystallization trials, are also fully integrated in the discipline. The ICCBMs were pivotal in promoting essential synergies for more rational design of crystallization experiments and a better understanding and control of protein crystal growth processes. As in other scientific disciplines, the choice of appropriate models is essential for deciphering the fundamentals. In the case of crystallogenesis, the small proteins lysozyme and thaumatin, still under investigation, provided such models. They were easily available, crystallize readily, and were inexpensive. Over the years, the crystal* Corresponding author. Phone: 33 (0)3 88 41 70 58. Fax: 33 (0)3 88 60 22 18. E-mail:
[email protected]. Web: http://www-ibmc.u-strasbg.fr/arn/ Giege/index_giege_fr.html. † Part of the special issue (vol 7, issue 11) on the 11th International Conference on the Crystallization of Biological Macromolecules, Québec, Canada, August 16–21, 2006 (preconference August 13–16, 2006). ‡ University of California Irvine. # UPR 9002, IBMC du CNRS & Université Louis Pasteur.
lizability of many other proteins, as well as of a few nucleic acids, viruses, and other multimacromolecular assemblies, was investigated. At this time, many features of macromolecular crystallization, which were previously mysteries, are understood, and the discipline has achieved an advanced degree of maturity. It has provided innumerable benefits to structural biology. It is the objective of this brief survey to show how the ICCBMs contributed to this development.
2. From 1985 to 1989: ICCBM-1 to 3 The first of what were later to be called the International Conferences on the Crystallization of Biological Macromolecules (ICCBM) was organized by Robert Feigelson and Alex McPherson at Stanford University in Palo Alto, California, between August 14 and 16, 1985.3 It was titled the First International Conference on Protein Crystal Growth and attracted 140 participants from 8 different countries. The meeting was organized in less than six months with little support and was unable to offer any to speakers. Nonetheless, the level of enthusiasm more than compensated for the lack of resources. The conference was intended as a singular event; there was no expectation that others might follow. The first conference was inspired by the interplay of two powerful market forces: the burgeoning demand of the molecular and structural biology community for more macromolecular X-ray crystallographic results and the virtually unlimited supply of important, low abundance proteins that could then be provided by recombinant DNA technology. There were two other forces that drove the conference as well: the expanding search of the community of conventional, small molecule crystal growth scientists for new areas of endeavor and novel challenges, and the increasing involvement of macromolecular crystal growth in the United States and European space programs. From the preface of the proceedings of that first meeting we find the following, written by Professor Feigelson: “There was
10.1021/cg700683a CCC: $37.00 2007 American Chemical Society Published on Web 10/26/2007
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a large, worldwide community of protein crystallographers working on protein crystal growth and they had not previously met as a group to discuss problems of mutual interest nor had any interaction with the small molecule crystal growth community. It was my intention, therefore, to bring together both recognized experts and other workers from the field of protein crystal growth to an environment conducive to in depth discussions on the current state and future prospects for protein crystallization, and at the same time, introduce them to some of the more prominent members of the small molecule crystal growth community to help stimulate a much needed dialogue between them. The latter undertaking being somewhat unusual and not without a certain amount of risk.” The objectives were not entirely achieved, language differences persisted, but perspectives were changed and channels were opened. Horizons were expanded and ideas were exchanged. Scientists whose thoughts and discoveries would be felt far beyond this first meeting were persuasive and provocative in their presentations. These included scientists with biology focused interest, such as Michael Garavito, Steve Durbin, George Feher, Walter Littke, Alexander McPherson, Richard Giegé on the one side, and people with backgrounds in crystal physics and/or chemistry, such as Paul Shlichta, William Tiller, Roger Davey, and Peggy Etter on the other. Of particular prominence, and deserving of special note, was Franz Rosenberger who mesmerized the entire assembly with his reconciliation of macromolecular observations with small molecule crystal growth theories and physics.4 Rosenberger’s dominant influence on the field, and its beneficial consequences were to be felt for the next decade and a half. The keynote address was given by Charlie Bugg, who outlined the scientific problem with great acuity: There have been major advances in the technology involved in determining protein crystal structures, once suitable crystals are available. The major bottleneck in the widespread application of protein crystallography is the difficulty in obtaining large, high-quality crystals of biological macromolecules for structural analysis. Most macromolecules are difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. Interestingly, and in spite of the progress that has been made over the past 20 years, those words remain true today. The first meeting was largely dominated by one particular interest group comprised of those who were National Aeronautics and Space Administration (NASA) or European Space Agency (ESA) investigators, or prospective investigators. Indeed, the support of the space agencies, and their funding of early investigators in the protein crystal growth community were crucial to the development of the entire field. Because of this, we find that at this first meeting, and at many subsequent meetings, the tone was less biological, and tended toward detailed observation and rigorous explanation of crystal growth phenomena in strict physical–chemical terms. At the same time, this was paired with an ongoing effort to explore and propagate new methods and techniques of an entirely practical nature. Thus, the ICCBM meetings have always been founded on two major lines of endeavor: producing crystals of macromolecules for X-ray diffraction analysis and understanding the physical– chemical bases for the mechanisms of protein, nucleic acid, and virus crystal growth. Appropriately, the conference was capped by a delightful banquet at the Paul Mason Winery on a mountaintop overlooking the shimmering lights of Silicon Valley. The success of this first conference was unanimously acknowledged, and the overall
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level of enthusiasm and optimism of all in attendance was markedly raised. By consensus, it was agreed to hold another such meeting, and the French participants stepped forward to carry the flag two years hence. The interval between the first and second conferences was marked by a number of developments important to the protein crystal growth community. These included an increasingly active microgravity crystallization program, with more frequent space flight opportunities for interested investigators. At the same time, recombinant DNA technology and the production of low abundance, high significance proteins were making unprecedented strides. In parallel were major advances in the development of high intensity synchrotron X-ray sources and rapid twodimensional X-ray detectors. These all conspired to emphasize the stature and significance of macromolecular crystal growth and to propel it forward. The Second International Conference on Protein Crystal Growth was splendidly organized by Richard Giegé with the help of two co-organizers, Arnaud Ducruix and Juan FontecillaCamps at the Bischenberg Conference Center just outside of Strasbourg, France, between July 19 and 25, 1987.5 In every way grander and more expansive than the initial meeting in Palo Alto, the accommodations, cuisine, and wine guaranteed success from the beginning. It was attended by 217 scientists from 21 different countries, with many participants from across Europe joining the nucleus of Stanford participants. NASA and ESA continued their enthusiastic support, but they were now joined by other sponsors from both the public and private sector. The meeting was, in addition to its official title, also a FEBS Advanced Lecture Course, and students became a major constituent. The keynote lecture was delivered by Nobel laureate and crystallographic pioneer Dorothy Hodgkin who recounted the earliest days of the field and commented optimistically on its prospects for the future. Other old hands joined the assembly including Jan Drenth, Reuben Leberman, Roland Boistelle, Larry Sieker, Roberto Poljak, and Serge Timasheff. The conference now had individual sessions with specific topics in physics, solution properties, practical approaches, microgravity, and discussions of specific problematic macromolecular systems. There were 80 posters and presentations by many of the people who would later make major contributions to the field, such as Mitsuo Ataka, Terese Bergfors, Charlie Carter, Naomi Chayen, Michel Frey, Andrzej Joachimiak, and Pat Weber. Among the exciting presentations were those of Ada Yonath who described the first crystallization of ribosomal subunits,6 and Mark Pusey who visualized convection plumes swirling about growing lysozyme crystals.7 It was at this meeting that ideas began to emerge for genetically engineering proteins to crystallize better, that the physics of crystal growth assumed a prominent role, automation of crystallization experiments first appeared, and the full panoply of biological macromolecules from peptides to viruses was explored. It was at Bischenberg that the community came to realize and appreciate the spectrum of opportunities and challenges that followed from the Stanford meeting. Following the second conference, microgravity crystallization was in abeyance due to the Challenger spacecraft disaster, but study of the physics of protein crystal growth continued apace. New ideas for screening crystallization conditions using specific reagent combinations began to appear and propagate. The ideas brought into the mix by the small molecule community, both classical and novel, began to take hold. During this period of consolidation, physics came to the fore.
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It was at the third meeting held at the University of Maryland Conference Center near Washington, D.C., and organized by Gary Gilliland and Keith Ward, that the gathering assumed its current name. The Third International Conference on Crystallization of Biological Macromolecules (ICCBM-3) was held between August 13 and 19, 1989, and its size swelled to over 300 participants with extensive American representation.8 The scientific scope of the meeting was equal to the attendance, with 10 different sessions spanning the range of topics from physical chemistry to microgravity and practical laboratory problems. Even the history of protein crystal growth was addressed in the opening lecture.9 An entire session was devoted to membrane protein crystallization, as this area began to emerge from darkness and assume an important place in the constellation of biological problems. Light scattering investigations of nucleating protein solutions were prominent, as were talks on crystal growth in gels, and not only automated experimental setups, but automated observations as well. Enrico Stura introduced streak seeding, Maria Garber and Sergei Trakhanov from Russia spoke on the crystallization of ribosomes, and Madeleine Riès-Kautt described intriguing studies on ion effects. The social events included generous helpings of Southern BarB-Que and a leisurely evening cruise down the Potomac River, welcome opportunities to relax and reflect. Though scientifically intense, the enthusiasm for crystal growth science was undampened. Although now dominated by the macromolecular community, important contributions continued, principally in the physics of nucleation and growth processes, from the small molecule crystal growth community whose input continued to inspire new ideas and novel approaches. These came, as always, from Franz Rosenberger, but as well from Allan Myerson, Marie-Claire Robert, and Jim Baird among others.
3. From 1990 to 1999: ICCBM-4 to 7 Between ICCBM-3 and ICCBM-4, the geopolitical scene became strangely disordered and began a process of restructuring, but the macromolecular crystallization community remained cohesive. The number of physiologically, pharmaceutically, and industrially important proteins that had been crystallized, and whose structures had been solved, increased dramatically. The PDB, or Brookhaven Data Bank as it was called then, was expanding its holdings at an extraordinary rate, and this only served to whet the appetite of the molecular biologists for more. Crystallography was moving into high gear. ICCBM-4 was organized solely by Walter Littke, really the first explorer of protein crystallization in microgravity, at the University of Freiburg in Germany between August 18 and 24, 1991.10 Cultural events and cuisine again competed with science for attention and admiration, including several concerts at extravagant venues, the ornate city hall, a restored baroque church, all involving both modern and ancient instruments. In the end, however, science again was dominant, with a vast range of new observations, discoveries, and explanations, and the introduction of several powerful new techniques. The realization that crystal growth had to be painstakingly optimized for best results, and how to go about it, consumed an entire session. Sample preparation and purification, a perennial topic, was dealt with in numerous talks, as were specific areas of biological interest such as antibodies and protein–protein complexes. The unexpectedly difficult and contentious problem of assessing the quality of protein crystals and protein crystallization experiments was opened to discussion, a topic revisited numerous times in subsequent conferences, and a continuing question among microgravity researchers.
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Innovations and new ideas appeared in every session. Naomi Chayen and Patrick Shaw-Stewart introduced the use of microbatch crystallization under oil.11 McElroy spoke on the mutation of protein surface residues as an approach to enhancing crystallization.12 Peter Bennema continued the building of bridges between the macromolecular and small molecule crystal growth communities with a discussion of the application of chain theory to protein crystal growth.13 Dynamic light scattering was further advanced as a tool for predicting crystallization and detecting aggregation, by Alan D’Arcy14 and Bill Wilson.15 One particular presentation stood out as truly exceptional, and that was Steve Durbin’s description, amply illustrated by images, of his pioneering atomic force microscopy (AFM) studies of lysozyme crystals actively growing in their mother liquors.16 This talk introduced the community to AFM as a powerful tool, along with interferometry, for visualizing the mechanisms by which surface layers on actively growing protein crystals develop, and a way to measure the fundamental kinetic and thermodynamic parameters that govern the process. An interesting aspect of protein crystal growth to emerge between ICCBM-4 and ICCBM-5 was the appearance of a commercial component. Small companies, most prominently Hampton Research, were founded and began to produce kits for screening crystallization conditions. This development had a profound impact because it opened the field to everyone. Anyone, any molecular biologist or biochemist, who had a beloved protein could make an honest try to crystallize it, and if successful, overnight become a crystallographer. In parallel, automation and robotics were now available to enhance speed and reproducibility. And beyond even these innovations, crystallization databases were constructed to guide the crystal grower in the approaches he might take.17 Inarguably, the most relaxed of the conferences, or laid back as they say in Southern California, was ICCBM-5, which was organized by Enrico Stura and John Sowadski at the Catamaran Hotel on the shores of Mission Bay in San Diego, California.18 Salt water was an only step away from the conference room, and most lunches and dinners were served on the beach patio. In spite of the temptations, science nonetheless prevailed. ICCBM-5 was sponsored by the National Institutes of Health (NIH) to also serve as a membrane protein crystallization workshop, and this served to introduce many in the membrane community to the conferences and to attract many noted authorities in the field. In addition, the conference was preceded by a workshop on the growth of two-dimensional macromolecular crystals for electron microscopy studies, until then, a much-neglected area. Membrane protein crystallization had clearly emerged as a major endeavor and an area of intense interest to the broad structural biology discipline, as well as to the biotechnology and pharmaceutical industries. The meeting, as with prior conferences, was well attended, with nearly 300 participants from all over the world, drawn by the sunshine, the sand, and the science. At ICCBM-5, molecular biology, and its applications in crystal growth research, returned to center stage and took back the emphasis from physics and observation. The keynote lecture was given by Richard Giegé with a focus on the biochemistry, preparation, and purification of proteins and their complexes, and their implications for crystal growth. This was echoed throughout the conference by numerous presentations of strongly biochemical focus. Practical aspects of crystallization were treated with more reverence. The problem of finding initial, successful conditions through screening procedures was addressed by many speakers, including among others, Charlie Carter19 and Bob Cudney.20
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In part because of the continuing interest in microgravity crystallization, and the development of means to simulate its effects, crystal growth in gels was an active area of research. Juan-Ma Garcia-Ruiz and Abel Moreno introduced their gel acupuncture technique,21 now in practice as a counter-diffusion procedure to explore a broad area of the crystallization phase diagram. A seminal presentation was that of Bill Wilson who for the first time introduced explicitly the concept of using the second virial coefficient of a mother liquor, measured by static light scattering methods, to predict successful crystallization conditions.22 This approach had a profound effect in that it inspired innumerable, subsequent investigations, and reawakened an interest in the physical chemistry of concentrated protein solutions and their similarities to colloids. There was another new and welcome addition. For the first time, an ICCBM had an array of commercial exhibitors and vendors for the protein crystal growth community. ICCBM-6 took place between November 12 and 17, 1995, at the Aster Plaza in Hiroshima, Japan. The four plenary lectures (Franz Rosenberger, Alex McPherson, Hartmut Michel, and Noriyoshi Sakabe) harmoniously blended physics, biology, and crystallography. The cochairs of ICCBM-6, Tamaishi Ashida and Hiroshi Komatsu, in the preface to the Proceedings,23 noted that the program of the conference “reflected the growing realization by structural biologists that the practice of macromolecular crystal growth can benefit from applying the physicochemical principles underlying small molecule crystallization”. Franz Rosenberger, in his lecture, critically analyzed the understanding the community held in 1995 regarding the nucleation and crystallization of globular proteins and detailed the many remaining gaps.24 Importantly, his talk was one of the first examples of a crystal growth physicist using biochemical results from his own laboratory to explore a key aspect of crystallization, namely, the role of impurities. His bold example was widely followed. The role of impurities was also examined by Alex McPherson who presented an impressive gallery of AFM images, which demonstrated how foreign materials (dust particles, microcrystals. . .) may be incorporated in growing protein and virus crystals.25 In another keynote lecture, Nobel laureate Hartmut Michel emphasized, with pertinent examples from his laboratory, that purification, as much as crystallization, was the rate-limiting step in membrane protein structural biology. This perspective is no less valid for many other macromolecules, and the series of ICCBM conferences consistently emphasized purification as an integral part of crystallogenesis. The Hiroshima conference, to its great good fortune, provided the opportunity for Alex Chernov, one of the intellectual fathers of crystal physics, to enter the circle of biocrystallogenitors. By 1995, diffraction data collection using synchrotrons was an integral part of X-ray crystallography. It had become a tool essential to crystallographers if they were to meet the expectations of structural biologists eager to study macromolecular crystals with large unit cells, visualize proteins and nucleic acids at ultra high resolution, and investigate molecular dynamics using time resolved Laue methods. Synchrotron radiation and advanced detector technology were discussed in detail by Noriyoshi Sakabe, one of the world’s foremost authorities, from Tsukuba, along with the recent Japanese success in solving the imposing structure of cytochrome c oxidase, a membrane protein of exceptional importance. A number of topics novel to protein crystallography were addressed as well at Hiroshima. These included Ostwald ripening, improved crystallization methods under oil, and
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ribosome crystallography. Scientific and social interactions flourished at the conference center and during a memorable excursion to the island of Miyajiwa, one of most beautiful parks in Japan, especially in autumn when decorated with the kaleidoscopic colors of maple leaves. Organization of the scientific program, as well as cultural events during ICCBM-6, were flawless. Hiroshima provided abundant inspiration to many of the participants who enthusiastically anticipated the next ICCBM conference. Following the conference in Japan, ICCBM-7 moved to Granada and mysterious Andalusia in the south of Spain. The conference was organized by Juan-Ma Garcia-Ruiz from May 3 to 8, 1998.26 Besides refined views on the physics of protein solution properties, nucleation, and crystal growth, the Granada conference highlighted methodological trends, in particular, the in-house counter-diffusion crystallization method.27 It also focused on advances in the observation of crystal growth processes, and their management, and new aspects of crystal quality assessment. Interesting correlations were drawn between macromolecular crystallization and the crystallization of colloids, and the topic of large statistical fluctuations in concentrated protein solutions and their relevance to nucleation was enthusiastically debated. An entire session devoted to microgravity was marked by the presentation of Mach–Zehnder phase shift interferograms visualizing concentration gradients around growing lysozyme crystals.28 A poster describing crystallographic improvement for microgravity versus terrestrial grown lysozyme crystals and correlation with the exclusion of specific protein impurities, attracted particular attention.29 The 1990s was a period when progress in the structural biology of RNA began to accelerate, and this became apparent in a focused session at ICCBM-7. Adrian Ferré D’Amaré argued that exhaustive screening of crystallization conditions alone, even with homogeneous RNA, does not ensure success. He developed the concept of general modules for RNA crystallization and illustrated his ideas with results on the ribozyme from hepatitis delta virus, which could not be crystallized except when modified to bind the U1A protein.30 In another area, Ehud Landau gave a remarkable talk on the use of lipidic cubic phases for membrane protein crystallization, a promising tool advanced and elaborated upon by Martin Caffrey at ICCBM-11 in Québec.
4. Biocrystallogenesis in the 21st Century: ICCBM-8 to ICCBM-10 From the shores of Mission Bay, site of ICCBM-5 in 1993, to the beaches of the Gulf of Mexico, ICCBM-8 took place between May 14 and 19, 2000 at the Sandestin Resort and Conference Center on the northwest coast of Florida.31 The conference was cochaired by Larry de Lucas, the crystallographer–astronaut, and Alex Chernov, the distinguished authority on the physics of crystal growth. ICCBM-8 placed particular emphasis on the physics and microgravity aspects of crystallogenesis but by no means neglected more biologically inspired topics. At the Sandestin conference, the idea of practical training was reprised, and a one-day workshop was directed by Alex McPherson on the day preceding the meeting. This became an event that accompanied all subsequent gatherings. The Florida conference opened with a session on highthroughput crystallization, a topic pursued in greater depth at subsequent ICCBMs. A novelty at the Sandestin conference was a session focusing on crystallization strategies from the perspective of screening and scoring of results. Lysozyme continued as the dominant model for the physical aspects of crystal growth, exemplified at ICCBM-8 by a talk on the control of its
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nucleation.32 Also noteworthy were the visualization by video and interferometric images of a supersaturation wave as seen in microgravity,33 examples of neutron crystallography with large crystals grown in microgravity,34 and results from improved crystals grown under high magnetic field.35 The atmosphere during ICCBM-8 was relaxed and the Sandestin Beach Resort provided ample opportunities to discuss science in an idyllic setting of golf greens and white sand beaches. It was attended by almost 350 participants from 24 countries. ICCBM-9 was organized by Rolf Hilgenfeld and held between March 23 and 28, 2002 in the University town of Jena, in Thuringen, Germany.36 It enjoyed an extremely broad representation of the crystallogenesis community with 420 participants,2 nearly three times that of the first conference in 1985. Structural genomics and the requisite high-throughput methods, automation, and other methodological and instrumentation aspects were central, as were considerations of the proper statistical analysis of crystallization data and the creation of databases. The Jena conference popularized the Granada box for counterdiffusion crystallization37 and advanced the full automation of crystallization in structural biology. Enthusiastic lectures came from many structural genomics centers including, to name but one, the Tuberculosis Structural Genomics Consortium in California.38 In addition to these application-focused interests, Jena also contributed to a greater understanding of the physical–chemical aspects of protein crystallization. This was exemplified by three presentations, among numerous others. The first dealt with the effects of anions on lysozyme crystallization,39 and the profound, specific effects of cations on lysozyme solubility.40 In another talk, based on the results of SAXS measurements and thermodynamic considerations, Annette Tardieu discussed the respective advantages and disadvantages of monovalent salts and PEG as crystallizing agents.41 In the third presentation, Peter Vekilov described a retrograde solubility dependence of hemoglobin C on temperature. Thus, he could explain crystallization of this protein by an entropic contribution arising from the release of water molecules at lattice contacts.42 ICCBM-10 was organized in Beijing, China, June 5–8, 2004.43 The focus of the conference, as emphasized by chairman Zihe Rao, was on “new concepts, methods and technologies to make the crystallization of biological macromolecules more efficient”. High throughput crystallization for structural genomics, and the preparation and crystallization of complex macromolecules and macromolecular assemblies were major themes, as well as assessment of crystal quality by X-ray methods. At the Beijing conference, conventional protein crystallographers were more in evidence, with plenary lectures from, among others, Michael Rossmann, Jack Johnson, Wenrui Chang, Shigeyuki Yokoyama, Tom Blundell, Bi-Cheng Wang, and Joel Sussman. Their attendance was an impressive confirmation of the crucial importance of the field of crystallogenesis to the present and future challenges of structural biology. Other plenary lectures covered practical aspects of macromolecular crystallization, crystal quality assessment, and novel trends in data collection (Alex McPherson, John Helliwell, Noriyoshi Sakabe), as well as the physics of biocrystals and biocrystallization (Alex Chernov and Peter Wills). Some speakers discussed the proliferation of crystallization screening kits and its consequence that people rely too heavily on them at the expense of critical thought about the experiments themselves, a topic given further attention in quiet discussions among participants. Though crystallogenesis shared the stage with structural analysis, numerous new findings nonetheless appeared that
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deserve particular note. These included measurement of surface height fluctuations on growing lysozyme crystals,44 and an explanation of high-pressure-induced ordering of cubic crystals of icosahedral cowpea mosaic virus.45 The crystallization of thaumatin at high pressure (150 MPa) was also recounted, as was a subsequent rearrangement of the hydration shell of the crystalline protein.46 The conference provided abundant opportunities to exchange ideas during the sessions, as well as the many social and cultural events. The banquet was extraordinary in many ways, especially the exquisite cuisine and the music played on ancient Chinese instruments. It, furthermore, provided an occasion to recognize many young scientists for their excellent poster presentations. For those who wished to see more of China, outings to the Great Wall and the terracota armies at Xian followed.
5. Dominant Themes of the ICCBMs Several themes were recurrent through the ICCBM conferences. One was purity, and related to it, impurities in protein crystallization as examined from the perspectives of both biochemistry and physics. The mechanisms and physics of protein crystal growth processes consistently occupied a central, and often dominant role in the conferences. Even when the field was in its infancy, in the 1980s, physics developed rapidly and became a major new activity in a number of traditional protein crystallization laboratories. ICCBM conferences generally devoted particular attention to macromolecular solution properties and the effects of ions, solVents, and cosolVents, to crystal perfection and defect structure, to physical processes related to micrograVity, and to comparisons with the solution growth of small molecules. At the same time, ICCBM conferences provided extensive coverage of the practical aspects of protein crystallization, often neglected and underappreciated by structural biologists, and they included salient biological issues such as nucleic acid and membrane protein crystallization. As a consequence of these efforts, advanced crystallization procedures and new reagents were developed. In what follows, we highlight several noteworthy contributions that significantly advanced the field. 5.1. Purity and Impurities. The influence of purity and impurities on protein crystal growth were major themes at every conference, both from the standpoints of biochemistry and physics. These are broad topics of multiparametric nature that impact the macromolecule itself, as well as a range of crystal properties such as mosaicity and the cryogenic characteristics of macromolecular crystals. ICCBMs led to the concept of conformational purity of macromolecules, and to the conclusion that chemical and conformational micro-heterogeneities in protein or RNA molecules were likely the most detrimental types of impurities in crystallization. On the other hand, unrelated macromolecules present in samples often do not significantly interfere with crystal lattice formation. Finally, theoretical considerations and advanced observation techniques provided access to the pathways by which impurities affect crystal growth mechanisms. Typical examples of the diversity of this theme at ICCBMs are illustrated by the following references.1,24,25,29,47–54 5.2. Solution Properties. Two lines of research, the first being the solubility of proteins in crystallizing media, and the second, the association behavior of proteins under precrystallization conditions, were cornerstones of all ICCBMs. A number of groups contributed probing studies on the solubility of proteins, frequently on the model protein lysozyme, in the presence of various ions or nonionic precipitants, and as a function of temperature or pH. These investigations culminated
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at ICCBM-9 in Jena with about a dozen related conference papers.36 With regard to precrystallization, it became evident that proteins must not be allowed to aggregate or denature in the mother liquor used for crystallization. This followed a renewed interest in light scattering methods to monitor prenucleation events. After the pioneering studies in George Feher’s laboratory on monitoring lysozyme crystallization by dynamic light scattering (DLS),55 several groups had used the method to investigate for crystallogenesis purposes, the prenucleation of insulin and canavalin56 and of amylase.57 It was also used as a tool to diagnose and optimize crystallization of aminoacyltRNA synthetases and elongation factor,58,59 and to study a series of proteins of interest to pharmaceutical research.14 The contributions of Bill Wilson, who proposed the osmotic second virial coefficient as a predictor of crystallization perceptive in defining the crystallization slot where crystals might be expected to grow,22 and further delineated the correlation between this coefficient and protein solubility,60 were instrumental in providing greater insight into the process of crystallization. In addition to DLS, small-angle X-ray and neutron scattering (SAXS and SANS) yielded additional important information on the interaction between proteins prior to crystallization. For example, SAXS was used to identify an increase in attractive interactions with increasing salt concentrations during lysozyme crystallization,61,62 and SANS was employed to decipher the crystallization properties of halophilic malate dehydrogenase in a ternary NaCl-MPD-H2O system that occupied a unique region of the phase diagram.63 5.3. Microgravity. Despite skepticism in some quarters, to some extent justified, macromolecular crystallization attempts under microgravity and analysis of data originating from such experiments, were nonetheless crucial in the development of biocrystallization as a mature scientific discipline, a discipline that has become indispensable to molecular and structural biology. From the pioneering experiments presented at ICCBM164 to ICCBM-10, two decades of microgravity experiments were carried out and analyzed.65 Driven by the microgravity projects, the ICCBM series generated a wealth of results that served to promote an understanding of crystal growth under diffusive transport regimes, and it fostered the development of numerous, novel approaches to the crystallization of macromolecules under conditions of reduced convection. Crystallization in gels was thoroughly investigated,66–68 and the value of counter-diffusion crystallization methods was clearly demonstrated.33 From a more operational, practical standpoint, space agencies were pillars of support for many protein crystal growth laboratories, and for the early ICCBMs. 5.4. Toward Better Crystals and Crystal Perfection. The topics of protein expression and purification as they pertained to improved crystals were consistently featured at ICCBMs,69,70 as was the question of objective and quantitative measures of crystal quality. John Helliwell was particularly influential in promoting studies on protein crystal perfection, defects, and radiation damage using highly collimated synchrotron radiation.71 The correlation between mosaicity and crystal defect structure took some time to be understood but became increasingly evident when X-ray topographic images became available and profiles of individual X-ray reflections could, with care, be recorded. Images frequently revealed the existence of internal dislocations and discontinuities in crystal lattices that, otherwise, showed no evidence of defects. Analyses of protein crystal quality and perfection are exemplified by the following references.52,72–76
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5.5. Biological Issues. The biology of crystallization systems was always a core component of ICCBM conferences and provided the inspiration and motivation to confront persistent challenges. The examples that follow convey only a flavor of the variety of macromolecules investigated, and of which crystals were sought and successfully produced: DNA-binding repressor,77 HIV reverse transcriptase,78 proteins of interest to pharmacology,14 retinoic acid binding proteins,79 membrane proteins,80 hormone binding proteins,81 ribosomes,82 and viruses.45
6. Novel Trends Highlighted in ICCBMs The ICCBM series proposed noVel crystallization methods to structural biologists, as exemplified by crystallization in gels,66 in levitated drops,83 under oil,11 by gel acupuncture,21 under magnetic field,84 by counter-diffusion,27,37 under high pressure,85 under an external electric field,86 with nucleation initiation on mica plates,87 and by stirring.88 Some of these methods have already become popular in laboratories throughout the world, such as crystallization under oil or in the diffusive transport regime. Others await further validation. Highthroughput approaches for structural biology and crystallography, renamed crystallonomics by Joe Ng at ICCBM-10 in Beijing, represent another novel trend. A necessity to miniaturize crystallization experiments became increasingly evident,89–91 culminating in the development of microfluidic technologies, a theme that first appeared at ICCBM-792 and became prominent at IBBCM-11. Microfluidics was a topic at ICCBM-9 in Jena as well, where Piet Abrahams discussed advances in protein nanocrystallography. Novel crystallization strategies based on chemical considerations, for example, combinatorial approaches,93 use of intelligent screens for membrane proteins,94 and rational use of additives, such as MPD,95 have more recently appeared. Finally, ongoing attempts to understand the physics of membrane protein crystallization and to design rational approaches to membrane protein crystallization deserve attention. By DLS, and other optical techniques, it was demonstrated that crystallization of bacteriorhodopsin solubilized with the surfactant octyl-glucoside is correlated with the interaction of the surfactant with the hydrophobic protein, the concomitant micelle structure, and the phase behavior.78 This was just one of the many investigations of significance to membrane protein crystallization. Together, these trends have offered new approaches to structural biologists for the solution of difficult crystallization problems, and they opened new avenues for innovative crystallogenesis research. 7. Rationality versus Serendipity In the concluding remarks to ICCBM-2, Jan Drenth remarked that the purpose of the meeting was to try to remove the magic in protein crystallization and give a sound theoretical basis to a field that was in the 1980s at the same level as chemistry in medieval time.96 This expectation has now largely been fulfilled, although all difficulties have not yet been surmounted. Obstacles and questions continue to challenge the field, and bottlenecks still exist. Fortunately, however, serendipity, that constant companion of crystal growers, occasionally rewards and inspires investigators of crystallogenesis. Macromolecular crystals, to the eyes of molecular biologists and crystallographers, exceed in beauty even the brightest and most awesome jewels. In future ICCBMs, it may be hoped that new strategies will be devised, and additional tactics be developed, to better direct and channel serendipity, and that the remaining ambiguities will ultimately be resolved.
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Acknowledgment. This work was supported by the French Centre National de la Recherche Scientifique (CNRS).
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CG700683A
