CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 11 2124–2125
PerspectiVes Good Crystals, Still a Challenge for Structural Biology† Sheng-Xiang Lin,*,‡,§ Alexander McPherson,| and Richard Giegé⊥ Oncology and Molecular Endocrinology Laboratory, CHUL Research Center (CHUQ) and LaVal UniVersity, 2705 BouleVard Laurier, Sainte-Foy, Québec G1V 4G2, Canada, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, China, Department of Molecular Biology and Biochemistry, UniVersity of California, IrVine, California, 92697, and UPR 9002, IBMC du CNRS & UniVersité Louis Pasteur, 15 rue René Descartes, F-67084 Strasbourg Cedex, France ReceiVed August 7, 2007; ReVised Manuscript ReceiVed August 15, 2007
The International Conference on the Crystallization of Biological Macromolecules (ICCBM-11) was held in late August 2006 at Laval University in Quebec City with the support of the research center of “the Centre Hospitalier de l’Université Laval”. It was held at a time when the field of biological crystallogenesis was entering a new phase, one that emphasizes the creation and development of novel tools and strategies to grow crystals more reliably for high resolution studies. The conference was attended by more than 200 scientists from 26 countries and provided an opportunity for discussion of the present state of the discipline from the viewpoints of both academia and industry. It encompassed all aspects of the physics of protein crystal growth, practical applications, new crystallization strategies, structural genomics, and robotics and provided many examples where crystallogenesis proved the essential means of answering basic biological questions. Preparation of high-quality macromolecular crystals has long been a persistent bottleneck to the greater application of X-ray crystallography in structural biology. Today, however, inspection of the Protein Data Bank (PDB, www.rcsb.org), with its impressive number of structures, would seem to indicate that crystallization difficulties have been largely overcome. This is true to some extent and is due to advances in sample preparation, screening methods, and automated crystallization technologies. A better understanding of the physics of the protein crystal growth process has also contributed to more rational approaches to protein crystallization. Scientific results reported at previous ICCBMs, summarized at the opening of ICCBM-11 (A. McPherson and R. Giegé), have, without question, contributed substantially to these advances. Many difficult challenges still † Part of the special issue (Vol 7 issue 11) on the 11th International Conference on the Crystallization of Biomacromolecules, Quebec, Canada, August 16–21, 2006 (pre-conference August 13–16, 2006). * Corresponding author. E-mail:
[email protected] (S.-X.L.);
[email protected] (A.M.);
[email protected] (R.G.). ‡ CHUL Research Center (CHUQ) and Laval University. § Visiting sceintist at Shanghai Institutes of Biological Sciences, China. | University of California, Irvine. ⊥ Université Louis Pasteur & IBMC du CNRS.
exist nonetheless, for example, in the crystallization of membrane proteins, large biomolecular complexes, nucleic acids, and other macromolecules with unusual physicochemical properties and in obtaining crystals for high-resolution crystallography and neutron diffraction. With regard to macromolecular physics, the relative importance of factors affecting the crystallization process, the role of impurities, crystal defects, microgravity, and numerous other effects is not fully understood. As with previous ICCBM gatherings, the physics of protein crystal growth received considerable attention. P. Vekilov (Houston) presented unexpected results on the mechanism of nucleation and growth of insulin crystals, novel in protein crystallization, where the viscosity of the crystallizing samples plays a critical role. A Japanese team exploring the mechanical properties of lysozyme crystals showed their connection with elasticity and characterized crystal imperfections.1 Particularly impressive were the results of two other Japanese scientists from Tohoku University at Sendai. They presented new instruments and a host of intriguing observations on molecular diffusion on crystal surfaces by advanced confocal microscopy and exquisitely sensitive interferometric approaches to analyze growth step advancement and growth mechanisms.2,3 M. Pusey (Huntsville) spoke on the development and use of fluorescent labels for identifying protein crystals. Likewise, the second virial coefficient and phase diagrams were explored as predictors of protein crystallizability by several groups from Canada, France and the USA (e.g., ref 4). In keeping with the current, intense interest in structural genomics, robotic approaches were described by several speakers, among them C. Betzel (Hamburg), K. Harlos (Oxford), and J. Luft (Buffalo). The development and value of crystallization databases were explored by H. Einspahr and others. Microfluidics also emerged as a subject of increasing interest, as protein samples decrease in size. Fluidigm Corporation presented recent advances to their system, and novel microfluidic devices were described by J.-C. Poulsen (Copenhagen) for the determination of protein precipitation boundaries and by C. Sauter (Strasbourg) for screening and optimization of crystallization conditions, as
10.1021/cg700745r CCC: $37.00 2007 American Chemical Society Published on Web 10/26/2007
Perspectives
well as for use in crystallographic analysis. A nice illustration of this technology was a demonstration of nucleation and growth decoupling (S. Fraden, Brandeis University). J. Ng and colleagues (Huntsville) had made a compelling argument in their presentation that small laboratories can do structural genomics. Several novel crystallization methods, or methods not generally familiar to structural biology laboratories, were convincingly advocated and illustrated by structural applications. Several milestones deserve attention. Particularly appealing is the method using femto-second laser irradiation or solution stirring to trigger forced nucleation at low supersaturation.5 This yielded high quality crystals of a tRNA modification enzyme in complex with its tRNA substrate6,7 and of several other proteins including a membrane protein. Counterdiffusion crystallization is wellknown by the physicochemists of crystal growth, but not widely by structural biologists. J.-M. Garcia-Ruiz (Granada) presented the fundamentals of the method8 and convinced the attendees of ICCBM-11 of its great potential. By definition, counterdiffusion works best in convection-free environments that are favored in thin capillaries, in gelled media and under microgravity. Also notable was a novel electrocrystallization technique worked out by A. Moreno (Mexico) for the study of bovine cytochrome C. A new crystallization strategy based on the inclusion of small molecules in mother liquors was presented by A. McPherson (Irvine), along with experimental evidence of its utility. T. Bergfors (Uppsala) reviewed fundamental approaches and their practical application, whereas N. Chayen (London) described the use of a new substrate for the promotion of crystal nucleation. Two methods for membrane protein crystallization provided hope that this difficult field is becoming more tractable. M. Caffrey (Limerick) was very convincing when he explained how crystals grow in heterogeneous lipidic media constituting cubic and lamellar mesophases.9 The second method is of general use for membrane and soluble proteins and is based on molecular recognition principles. The protein to be crystallized is cocrystallized with a specially designed ankyrin repeat protein (ARP) selected using ribosome display.10 M. Grütter (Zürich) illustrated the technology with three successful examples of ARP–protein complexes leading to structure determination, namely, complexes with a kinase, a caspase, and a membrane protein. This ARP method contrasts with the more classical, although not often used, technology of protein cocrystallization with antibody fragments. The novel high-throughput method for antibody fragment production, based on phage display and developed at the University of Georgia, Athens, rejuvenates this crystallization strategy. From a more general perspective, production of recombinant proteins for crystallization purposes is often a drawback. B. Haze from the University of Alberta presented combinatorial strategies to overcome such difficulties. Post-translational modifications that can cause structural microheterogeneities in proteins are commonly considered harmful for crystallization. J. R. Mesters (Lübeck) presented and critically discussed several successful crystallizations of glycoproteins, thus giving hope for easier access to the structures of the modified proteins so important in the eukaryotic world.
Crystal Growth & Design, Vol. 7, No. 11, 2007 2125
The conference also provided a forum that showed how crystallogenesis efforts could be essential for high-resolution X-ray crystallography or neutron crystallography and for solving important structures. The latters include proteins essential in bacterial structural genomics, SARS coronavirus biology, gene translation, toxicology, and human endocrinology, reported at ICCBM11. ICCBM-11 represented, in many regards, a reawakening of the imagination in the number and quality of truly new and innovative approaches presented. It appears that the community clearly recognizes that traditional methods of protein crystal growth will not produce the breakthroughs necessary to meet the advancing needs of structural biology. The community is enthusiastically responding to the challenge in creative and novel ways. The meeting also represented the culmination of long efforts, particularly in the areas of physics and chemistry, which demonstrated once again that persistence and tenacity are indeed rewarded. Most encouraging was that many new faces were seen and voices heard at ICCMB-11, an event that clearly represents a turning point for the community. Finally, the conference gave the opportunity to consolidate the International Organization of Biological Crystallization (IOBC, http://www.iobcr.org/ index.html), whose role is to promote the crystallogenesis field, to elect N. Chayen (London) to its chair and decide that ICCMB12 will be organized in 2008 by A. Moreno in Cancun (Mexico). All these provide good cause for optimism that macromolecular crystal growth, both in principle and in practice, may soon be taken well in hand. Acknowledgment. We acknowledge the program and organizing committes of ICCBM11, the cochairs of the preconference workshop, Dr. J.-M. Garcia-Ruiz and Dr. J. R. Mesters, as well as Ms. M. Mazumdar for her excellent assistance. We are thankful for the support of the Canadian Institutes of Health Research (Institutes of Cancer Research and Genetics) for the conference.
References (1) Koizumi, H.; Tachibana, M.; Kojima, K. Phys. ReV. 2006, E73, 041910. (2) Sazaki, G.; Tsukamoto, K.; Yai, S.; Okada, M.; Nakajima, K. Cryst. Growth Des. 2005, 5, 1729–1735. (3) Dold, P.; Ono, K.; Tsukamoto, G.; Sazaki, G. J. Cryst. Growth 2006, 293, 102–109. (4) Zhu, D.-W.; Garneau, A.; Mazumdar, M.; Zhou, M.; Xu, G.-J.; Lin, S.-X. J. Struct. Biol. 2006, 154, 297–302. (5) Adachi, H.; Matsumura, H.; Niino, A.; Takano, K.; Kinoshita, T.; Warizaya, M.; Inoue, T.; Mori, Y.; Sasaki, T. Jpn. J. Appl. Phys. 2004, 43, L522–L525. (6) Numata, T. Acta Crystallogr., Sect. F 2006, 62, 368–371. (7) Numata, T.; Ikeuchi, Y.; Fukai, S.; Suzuki, T.; Nureki, O Nature 2006, 442, 419–424. (8) Garcia-Ruiz, J.-M. Methods Enzymol. 2003, 368, 130–154. (9) Cherezov, V.; Clogston, J.; Papiz, M. Z.; Caffrey, M. J. Mol. Biol. 2006, 357, 1605–1618. (10) Binz, H. K.; Amstutz, P.; Kohl, A.; Stumpp, M. T.; Briand, C.; Forrer, P.; Grütter, M. G.; Plückthun, A. Nat. Biotechnol. 2004, 22, 575–582.
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