METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Preface Five years ago, Academic Press published parts A and B of volumes of Methods in Enzymology devoted to Macromolecular Crystallography, which we had edited. The editors of the series, in their wisdom, requested that we assemble the present volumes. We have done so with the same logical style as before, moving smoothly from methods required to prepare and characterize high quality crystals and to measure high quality data, in the first volume, to structure solving, refinement, display, and evaluation in the second. Although we continue to look forward in these volumes, we also look resolutely back in time by having recruited three chapters of reminiscence from some of those on whose shoulders we stand in developing methods in modern times: Brian Matthews, Michael Rossmann, and Uli Arndt. A spiritually similar contribu- tion opens the second volume: David Blow’s introduction to our Phases section has his personal reflections on the impact that Johannes Bijvoet has had on modern protein crystallography. In the earlier volumes, we foreshadowed a time when macromolecular crystallography would become as automated as the technique applied to small molecules. That time is not quite upon us, but we all feel rattling of the windows from the heavy tread of high-throughput synchrotron-based macro- molecular crystallography. As for the previous volumes, we have tried to provide in this volume sufficient reference that those becoming immersed in the field might find an explanation of methods they confront, while hopefully also stimulating others to create the new and better methods that sustain intellectual vitality. The years since publication of parts A and B have seen amazing advances in all areas of the discipline. Super high brightness synchro- tron sources (Advanced Photon Source in the United States, European Syn- chrotron Radiation Facility in Europe, and Super Photon Ring-8 in Japan) are producing numerous important results even while the older sources are increas- ing productivity. Proteomics and structural genomics have appeared in the lexicon of all biologists and have become vital research programs in many laboratories. In the spirit of the time, these chapters approach many of the methods that are pertinent to high-throughput structure determination. These are now robots for large-scale screening of crystal-growth conditions using sub-microliter volumes, which were accessible only in a few dedicated research laboratories a decade ago. Similarly, automation has begun to assume increas- ing roles in cryogenic specimen changing for data collection; many laboratories are building and beginning to use robots for this purpose. xiii The first and largest section of technical chapters dissects the cutting-edge methods for thinking about or accomplishing crystal growth, including theoret- ical aspects, using physical chemistry to understand and improve crystal dif- fraction quality, robotics, and cryocrystallography. The other large section addresses phasing. A profound shift has occurred with the growing appreci- ation that map interpretation and model refinement are inseparable from the phase problem itself. Various methods of integrating the two processes in automated algorithms constitute an important step toward realization of high-throughput. More importantly perhaps, they improve the resulting struc- tures themselves. New algorithms for representing the variance parameters have come into wider practice. The database of solved macromolecular structures has grown to the point where its statistical properties now afford impressive insight and can be used to improve the quality of structures. Concurrently, simulation methods have become more accessible, reliable, and relevant. The validation process is there- fore one that impacts a widening sphere of activities, including homology modeling and the presentation and analysis of conformational, packing, and surface properties. Many of these are reviewed in the concluding chapters. We take little credit, either for the quality of the volume, which goes to the chapter authors, or for comprehensive coverage of competing methods. We will happily accept blame for mistakes and omissions. Academic Press has remained supportive and helpful throughout the long and trying process of completing this job, earning our sincere appreciation. Charles W. Carter Robert M. Sweet xiv preface Contributors to Volume 374 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Jan Pieter Abrahams (8), Biophysical Structureal Chemistry, Leiden Institute of Chemistry, 2300 RA Leiden, The Netherlands Paul D. Adams (3), Lawrence Berkeley Laboratory, 1 Cyclotron Road, Berkeley, California 94720 Vandim Alexandrov (23), Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas, 77843 W. Bryan Arendall, III. (18), Depart- ment of Biochemistry, Duke University, Duke Building, Durham, North Carolina 27708 Nenad Ban (8), Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology, CH8093 Zurich, Switzerland Joel Berendzen (3), Biophysics Group, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Helen M. Berman (17), Research Callaboratory for Structural Bio- informatics, Department of Chemistry, Rutgers The State University of New York, Piscataway, New Jersey 08854 D. M. Blow (1), 26 Riversmeet, Appledore, Bideford, Devon EX39 1RE, United Kingdom Jose M. Borreguero (25), Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Axel T. Brunger (3), The Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology, and Neurological Sciences, Stanford Radiation Laboratory, Stanford University, 1201 Welch Road, Stanford, California 94205 Sergey V. Buldyrev (25), Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Kyle Burkhardt (17), Research Calla- boratory for Structural Bioinformatics, Department of Chemistry, Rutgers The State University ofNewYork, Piscataway, New Jersey 08854 Raul E. Cachau (15), Advanced Bio- medical Computer Center, Frederic, Maryland 21703 Stephen Cammer (22), University of California San Diego Libraries, 9500 Gillman Drive, La Jolla, California 92093 Charles W. Carter,Jr. (7, 22), Department of Biochemistry and Bio- physics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Zbigniew Dauter (5), Synchroton Radiation Research Section, NCI Broo- khaven National Laboratory Building, Upton, New York 11973 Feng Ding (25), Department of Biochemis- try and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 ix Eleanor J. Dodson (3), Department of Chemistry, University of York, Heslington York YO1 5DD, United Kingdom Nikolay V. Dokholyn (25), Department of Biochemistry and Biophysics, Univer- sity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Zukang Feng (17), Research Callabora- tory for Structural Bioinformatics, De- partment of Chemistry, Rutgers The State University of New York, Piscat- away, New Jersey 08854 Andra ´ s Fiser (20), Department of Bio- chemistry and Seaver Foundation Center for Bioinformatics, Albert Einstein Col- lege of Medicine, Bronz, New York 10461 Roger Fourme (4), Soleil (CNRS-CEA- MEN), Batiment 209d, Universite Paris XI, 91898 Orsay, Cedex France Mark Gerstein (23), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 Ralf W. Gosse-Kunstleve (3), Lawrence Berkeley Laboratory, 1 Cyclotron Road, Berkeley, California 94720 Dorit Hanein (10), The Burnham Institute, La Jolla, California 92037 Jan Hermans (19), Department of Bio- chemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Barry Honig (21), Department of Biochem- istry and Molecular Biophysics, Columbia University, New York, New York 10032 Thomas R. Ioerger (12), Texas A & M University, College Station, Texas 77843 Ronald Jansen (23), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 Andrzej Joachimiak (15), Structural Biol- ogy Sciences, Biosciences Division, Ar- gonne National Laboratory, Argonne, Illinois 60439 Jochen Junker (23), Max Planck Institut fur Biophysikalische Chemie, D37070 Gottingen, Germany Michel H. J. Koch (24), European Molecular Biology Laboratory, Ham- burg Outstation, D-22603 Hamburg, Germany W. G. Krebs (23), San Diego Supercom- puter Center, University of California San Diego, La Jolla California 92093 Victor S. Lamzin (11), European Molecu- lar Biology Laboratory, Hamburg Outstation, 22603 Hamburg, Germany Richard J. Morris (11), European Bioin- formatics Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SD, United Kingdom Garib N. Murshudov (14), Chemistry De- partment, University of York, Helsington York, YO1 5DD, United Kingdom Ronaldo A. P. Nagem (5), CBME Laboratorio Nacional de Luz Sincrotron and Instituto de Fisica Gleb Weataghin, Unicamp Caixa, CEP 13084-971 Campi- nas SP, Brazil Tom Oldfield (13), European Bioinfor- matics Institute, European Molecular Biology Laboratory, Wellcome Trust Genome Campus, Cambridge CB10 1SD, United Kingdom Miroslav Z. Papiz (14), Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, United Kingdom Anastassis Perrakis (11), Netherlands Cancer Institute, Department of Carcinogenesis, 1066 CX Amsterdam, The Netherlands x contributors to volume 374 Donald Petrey (21), The Howard Hughes Medical Institute, Department of Bio- chemistry and Molecular Biophysics, Col- umbia University, New York, New York 10032 Alberto Podjarny (15), Structural Biology Sciences, Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439 Igor Polikarpov (5), Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av Trabalhador, Saovarlense, 13560 Sao Carlos SP, Brazil Thierry Prange ´ (4), LURE (CNRS-CEA- MEN), Batiment 209d, Universite Paris XI, 91898 Orsay, Cedex France David C. Richardson (18), Department of Biochemistry, Duke University, Duke Building, Durham, North Carolina 27708 Jane S. Richardson (18), Department of Biochemistry, Duke University, Duke Building, Durham, North Carolina 27708 Jeffrey Roach (6), Department of Chemis- try and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Mark A. Rould (7), Department of Physi- ology, University of Vermont, School of Medicine, Burlington, Vermont 05405 James C. Sacchettini (12), Texas A & M University, College Station, Texas 77843 Andrej S ˇ ali (20), Mission Bay Genentech Hall, University of California at San Fran- cisco, San Francisco, California 94143 Celia Schiffer (19), Department of Bio- chemistry and Molecular Pharmacology, University of Massachusetts, Medical School, Worcesster, Massachusetts 01655 Marc Schiltz (4), LURE (CNRS-CEA- MEN), Batiment 209d, Universite Paris XI, 91898 Orsay, Cedex France Thomas R. Schneider (3, 15), Lehrstuhl fur Strukturchemie, Gottingen University D37077 Gottingen, Germany Eugene L. Shakhnovich (25), Department of Biochemistry and Biophysics, Univer- sity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 George M. Sheldrick (3), Lehrstuhl fur Strukturchemie, Gottingen University D37077 Gottingen, Germany H. Eugene Stanley (25), Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Dmitri I. Svergun (24), Institute of Crystallography Russian Academy of Sciences, 117333 Moscow, Russia Lynn F. Ten Eyck (16), National Partner- ship for Advanced Computational Infra- structure, San Diego Supercomputer Center, La Jolla, California 92093 Thomas C. Terwilliger (2, 3), Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Alexander Tropsha (22), Department of Medicinal Chemistry and Natural Products, University of North Carolina at Chapel Hill, Chapel Hill, North Caro- lina 27599 J. Tsai (23), Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas, 77843 Maria G. W. Turkenburg (3), Department of Chemistry, University of York, He- slington York YO1 5DD, United Kingdom Isabel Uson (3), Lehrstuhl fur Strukturch- emie, Gottingen University D37077 Got- tingen, Germany Patrice Vachette (24), LURE Bat. 209d, University Paris-Sud, F-91898 Orsay, Cedex France Iosif I. Vaisman (22), School of Computational Sciences, George Mason University, Manassas, Virginia 20110 Niels Volkmann (10), The Burnham Institute, La Jolla, California 92037 contributors to volume 374 xi Charles M. Weeks (3), Hauptman-Wood- ward Medical Research Institute, 73 High Street, Buffalo, New York 14203 John Westbrook (17), Research Calla- boratory for Structural Bioinformatics, Department of Chemistry, Rutgers The State University of New York, Piscataway, New Jersey 08854 Martyn D. Winn (14), Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, United Kingdom Kam Y. J. Zhang (9), Department of Struc- tural Biology, Plexxikon, Inc., Berkeley, California 94710 xii contributors to volume 374 [1] How Bijvoet Made the Difference: The Growing Power of Anomalous Scattering By D. M. Blow History Johannes Bijvoet (1892–1980) made pioneering contributions to the de- termination of noncentrosymmetric structures. He was the first to exploit the isomorphous replacement method to reveal a noncentrosymmetric structure, using isomorphous sulfate and selenate salts to determine the structure of strychnine on the basis of two projections. 1–3 In space group C2, the selenium atoms, one in each asymmetric unit, make a centrosym- metric array, and the structure factors of the heavy atoms (with appropriate choice of origin) are all real. The isomorphous difference then determines the real part of the strychnine structure factor, but the sign of the imaginary part of the structure factor is undefined. The best estimate of the strychnine structure factor is its real part. This leads to an electron density map in which the structure and its in- verse are superimposed, with symmetry C2/m. The structure of the strych- nine molecule was deduced by discarding one of each pair of atoms related by the mirror, using the same principles that Carlisle and Crowfoot 4 had used in separating the two images of the cholesteryl iodide molecule gener- ated by the ‘‘heavy atom’’ method. In both cases, the authors deriving their structure did not know which interpretation was a true representation of the molecule, and which was its inverted image. Bijvoet recognized that anomalous scattering could be used to identify the correct enantiomorph of a noncentrosymmetric structure. He wrote, 5 There is in principle a general way of determining the sign [of a phase angle]. We can use the abnormal scattering of an atom for a wavelength just beyond its absorption limit. It also becomes possible to attribute the d or l structure to an optically active compound on actual grounds and not merely by a basic convention. Nishikawa and Matsukawa 6 and Coster et al. 7 had observed departure from Friedel’s law 8 in diffraction from opposite polar faces of a zinc sulfide 1 C. Bokhoven, J. C. Schoone, and J. M. Bijvoet, Kon. Ned. Akad. Wet. 51, 825 (1948). 2 C. Bokhoven, J. C. Schoone, and J. M. Bijvoet, Kon. Ned. Akad. Wet. 52, 120 (1949). 3 C. Bokhoven, J. C. Schoone, and J. M. Bijvoet, Acta. Crystallogr. 4, 275 (1951). 4 H. C. Carlisle and D. M. Crowfoot, Proc. R. Soc. A 184, 64 (1945). 5 J. M. Bijvoet, Kon. Ned. Akad. Wet. 52, 313 (1949). [1] how bijvoet made the difference 3 Copyright 2003, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 374 0076-6879/03 $35.00 crystal. In a beautifully clear exposition of anomalous scattering effects, Bijvoet 9 drew on this example: Normal X-ray reflection does not detect any difference between one side [of the octahedral faces of a zinc blende crystal], a dull and poorly developed tetrahedron plane, and the other, a shining well-developed one. In this respect, it is less sensitive than the human eye. Coster, however, chose a radiation— L 1 radiation of gold—which just excites the K electrons of zinc. Now X-ray analysis not only detects a difference, but it concludes—and this is, of course completely impossible for the human eye—that it is the dull plane that has the zinc plane facing outwards. In 1951 Bijvoet and colleagues 10 observed the intensity differences between Friedel-related pairs of X-ray reflections from sodium rubidium tartrate crystals. These observed differences showed that the convention established by Emil Fischer to discuss the configuration of bonds at asym- metric carbon atoms, especially in sugars, by good chance represents the true three-dimensional enantiomorph of these molecules. This was a substantial achievement, but Bijvoet was looking much fur- ther ahead. His visionary paper in 1954 11 opens by mentioning the great successes of X-ray analysis that determined structures as compli- cated as those of sterols and alkaloids and that now approach the domain of Nature’s most complicated biochemical compounds, the proteins A flow chart (see Fig. 1) sets the agenda for structure determination for the next half-century. It shows how isomorphous substitution and anomalous scattering can determine phases for all noncentrosymmetric reflections. But there is a question mark. Bijvoet warns, 11 It has not yet been thoroughly investigated whether the small effect of the anomalous scattering will be measurable for a sufficient part of the reflections involved in a complete Fourier synthesis. How thrilled he would have been to know that tunable X-ray sources could produce such measurable effects that anomalous scattering could solve protein structures on its own! These methods began to be introduced in the last year of his life. 6 S. Nishikawa and R. Matsukama, Proc. Imp. Acad. Jpn. 4, 96 (1928). 7 D. Coster, K. S. Knol, and J. A. Prins, Z. Phys. 63, 345 (1930). 8 G. Friedel, Comptes Rendus 157, 1533 (1913). 9 J. M. Bijvoet, Endeavour 14, 71 (1955). 10 J. M. Bijvoet, J. F. Peerdeman, and J. A. van Bommel, Nature 168, 271 (1951). 11 J. M. Bijvoet, Nature 173, 888 (1954). 4 phases [1] Center of Symmetry PHASE DETERMINATION IN THE ISOMORPHOUS SUBSTITUTION METHOD (determination of amplitude sign) Heavy atom on center Algebraic amplitude addition (1936). Heavy atom out of center (a) Location of the heavy atom (Patterson analysis). (b) Algebraic amplitude addition (1939). No center of symmetry (determination of phase angle) (a) Location of the heavy atom. (b) Determination of the absolute value of phase angle from amplitude addition in vector diagram (1949). F B -F A (c) Synthesis of double Fourier and resolution (c') Determination of all phase signs by geometrical considerations. by anomalous scattering (19 ?) (d) Phase shift by anomalous scattering (1930). Determination of absolute configuration (1951). ∆f " ∆f " F B -F A Fig. 1. The Bijvoet presentation of phase determination in the isomorphous substitution method. Redrawn with permission from Nature 173, 888–891. Copyright 1954 Macmillan Magazines Limited. [1] how bijvoet made the difference 5 [...]... illustrated, denoted as 1 and 2 Data from reflection h are identified by a subscript plus symbol, and data from its Friedel mate Àh are denoted by a subscript negative symbol A possible value for FP is indicated as a dashed line, but because jFPj is not observable, its amplitude is unknown The open circle represents the structure factor if all atoms scattered normally included in the analysis, and the... applied to MAD, it is referred to as ‘‘pseudo-MIR’’ by Smith and Hendrickson The methods based on Karle’s approach,31–33 developed specifically for multiple wavelength studies, are called the ‘‘explicit’’ approach An important practical difference between the methods arises in identifying the positions of the anomalous scatterers In pseudo-MIR the observed Bijvoet amplitude difference directly provides coefficients... which individual atoms can be resolved, direct methods allow further refinement Many authors have reported excellent results using SAD A significant advantage is that because only one wavelength is used, the complication of resetting the apparatus to precisely defined wavelengths is avoided Data collection can proceed without interruption, so that radiation damage problems are greatly reduced, and more... fully automated fashion (SOLVE1) In addition, automated methods for refinement and model building with high-resolution data have been developed (wARP/ARP; Perrakis et al.2,3) The separate automation of structure solution and of model building and refinement presents the promise of full automation of the entire structure determination process from scaling diffraction data to a refined model The software... fully automated fashion (SOLVE1) In addition, automated methods for refinement and model building with high-resolution data have been developed (wARP/ARP; Perrakis et al.2,3) The separate automation of structure solution and of model building and refinement presents the promise of full automation of the entire structure determination process from scaling diffraction data to a refined model The software... Fleterick, and D A Agard, Acta Crystallogr D Biol Crystallogr 49, 429 (1993) 31 C Wilson and D A Agard, Acta Crystallogr A 49, 97 (1993) 21 30 phases [2] experimental data and prior knowledge about what the electron density map should look like are the most likely overall Until more recently the statistical foundation of density modification has been poorly developed.32–34 The procedure used to carry out density... clear how to weight the model and experimental phases in an optimal fashion Several methods have been devised to address this problem, including ‘‘solvent flipping’’35 and cross-validation,32,36 but neither of these approaches fully addresses the fundamental problem of the correlation between model and experimental phases.34 We have invented a method of density modification based on a statistical formulation... averaging.20,24–27 Additional density modification methods include histogram matching and phase extension,28 entropy maximization,29 iterative skeletonization,30,31 and iterative model building and refinement.2,3 The fundamental basis of density modification is that there are many possible sets of structure factor amplitudes and phases that are all reasonable based on the limited experimental data Those structure... is smaller, leading to a more tightly defined phase distribution This formulation22 is valid even when different types of anomalous scatterer exist, but usually one type of anomalous scatterer is assumed This method of phase determination was coded by L P Ten Eyck23 and by J E Ladner24 into a widely used program PHARE (now incorporating a maximum likelihood refinement procedure and called MLPHARE25) The... of Dickerson et al.13 for MIR cross-validation, in which one derivative is omitted from phasing and the others are used to calculate phases and a heavy 13 14 R E Dickerson, J C Kendrew, and B E Strandberg, Acta Crystallogr 14, 1188 (1961) T L Blundell and L N Johnson, ‘‘Protein Crystallography, ’ p 368 Academic Press, New York, 1976 [2] SOLVE and RESOLVE 27 atom difference Fourier for it We extended . Academic Press published parts A and B of volumes of Methods in Enzymology devoted to Macromolecular Crystallography, which we had edited. The editors of the series, in their wisdom, requested. Cambridge CB10 1SD, United Kingdom Garib N. Murshudov (14), Chemistry De- partment, University of York, Helsington York, YO1 5DD, United Kingdom Ronaldo A. P. Nagem (5), CBME Laboratorio Nacional de. 27599 ix Eleanor J. Dodson (3), Department of Chemistry, University of York, Heslington York YO1 5DD, United Kingdom Nikolay V. Dokholyn (25), Department of Biochemistry and Biophysics, Univer- sity