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Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

Nature Protocols volume 2, pages 1633–1651 (2007)Cite this article

Abstract

The preparation of protein single crystals represents one of the major obstacles in obtaining the detailed 3D structure of a biological macromolecule. The complete automation of the crystallization procedures requires large investments in terms of money and labor, which are available only to large dedicated infrastructures and is mostly suited for genomic-scale projects. On the other hand, many research projects from departmental laboratories are devoted to the study of few specific proteins. Here, we try to provide a series of protocols for the crystallization of soluble proteins, especially the difficult ones, tailored for small-scale research groups. An estimate of the time needed to complete each of the steps described can be found at the end of each section.

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Figure 1: Classification scheme for the results of a crystallization screen taken from ref. 75.
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References

  1. Berman, H., Henrick, K., Nakamura, H. & Markley, J.L. The worldwide Protein Data Bank (wwPDB): ensuring a single, uniform archive of PDB data. Nucleic Acids Res. 35, D301–D303 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Vainshtein, B.K. Modern Crystallography I Symmetry of Crystals, Methods of Structural Crystallography (Springer-Verlag, Berlin, 1981).

    Google Scholar 

  3. Chayen, N.E. Turning protein crystallisation from an art into a science. Curr. Opin. Struct. Biol. 14, 577–583 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. McPherson, A. Introduction to protein crystallization. Methods 34, 254–265 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. McPherson, A. Crystallization of Biological Macromolecules (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999).

    Google Scholar 

  6. Bergfors, T.M. Protein Crystallization: Techniques, Strategies and Tips (International University Line, La Jolla, CA, 1999).

    Google Scholar 

  7. Ducruix, A. & Giége, R. Crystallization of Nucleic Acids and Proteins, A Practical Approach. (IRL Press, Oxford, UK, New York, 1992).

    Google Scholar 

  8. Michel, H. Crystallization of Membrane Proteins (CRC Press, Boca Raton, 1991).

    Google Scholar 

  9. Iwata, S. Methods & Results in Crystallization of Membrane Proteins (IUL Biotechnology Series, La Jolla, CA, 2003).

    Google Scholar 

  10. Hunte, C., von Jagow, G. & Shagger, H. Membrane Protein Purification and Crystallization: A Practical Guide (Academic Press, New York, 2002).

    Google Scholar 

  11. Caffrey, M. Membrane protein crystallization. J. Struct. Biol. 142, 108–132 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Wiener, M.C. A pedestrian guide to membrane protein crystallization. Methods 34, 364–372 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Lundstrom, K. Structural genomics on membrane proteins: mini review. Comb. Chem. High Throughput Screen. 7, 431–439 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Asherie, N. Protein crystallization and phase diagrams. Methods 34, 266–272 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Chernov, A.A. Crystal growth and crystallography. Acta Crystallogr. A 54, 859–872 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Chayen, N.E. The role of oil in macromolecular crystallization. Structure 5, 1269–1274 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. McPherson, A. Protein crystallization in the structural genomics era. J. Struct. Funct. Genomics 5, 3–12 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Rupp, B. & Wang, J. Predictive models for protein crystallization. Methods 34, 390–407 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Rupp, B. High-throughput crystallography at an affordable cost: the TB structural genomics consortium crystallization facility. Acc. Chem. Res. 36, 173–181 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Sharff, A. & Jhoti, H. High-throughput crystallography to enhance drug discovery. Curr. Opin. Chem. Biol. 7, 340–345 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Morissette, S.L. et al. High-throughput crystallization: polymorphs, salts, co-crystals and solvates of pharmaceutical solids. Adv. Drug Deliv. Rev. 56, 275–300 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Stevens, R.C. High-throughput protein crystallization. Curr. Opin. Struct. Biol. 10, 558–563 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Chayen, N.E. Protein crystallization for genomics: throughput versus output. J. Struct. Funct. Genomics 4, 115–120 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Calderone, V. et al. The crystal structure of yeast copper thionein: the solution of a long-lasting enigma. Proc. Natl. Acad. Sci. USA 102, 51–56 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Derewenda, Z.S. & Vekilov, P.G. Entropy and surface engineering in protein crystallization. Acta Crystallogr. D Biol. Crystallogr. 62, 116–124 (2006).

    Article  PubMed  CAS  Google Scholar 

  26. Derewenda, Z.S. The use of recombinant methods and molecular engineering in protein crystallization. Methods 34, 354–363 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Giege, R. et al. The role of purification in the crystallization of proteins and nucleic acids. J. Cryst. Growth 76, 554–561 (1986).

    Article  CAS  Google Scholar 

  28. Zulauf, M. & D'Arcy, A. Light scattering of proteins as a criterion for crystallization. J. Cryst. Growth 122, 102–106 (1992).

    Article  CAS  Google Scholar 

  29. Thomas, B.R., Carter, D. & Rosenberger, F. Effect of microheterogeneity on horse spleen apoferritin crystallization. J. Cryst. Growth 187, 499–510 (1998).

    Article  CAS  Google Scholar 

  30. Rosenberger, F. Protein crystallization. J. Cryst. Growth 166, 40–54 (1996).

    Article  CAS  Google Scholar 

  31. Pusey, M.L. et al. Life in the fast lane for protein crystallization and x-ray crystallography. Prog. Biophys. Mol. Biol. 88, 359–386 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Pikal-Cleland, K.A. & Carpenter, J.F. Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase. J. Pharm. Sci. 90, 1255–1268 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Carpenter, J.F., Chang, B.S., Garzon-Rodriguez, W. & Randolph, T.W. Rational design of stable lyophilized protein formulations: theory and practice. Pharm. Biotechnol. 13, 109–133 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. McPherson, A., Malkin, A.J., Kuznetsov, Y.G. & Koszelak, S. Incorporation of impurities into macromolecular crystals. J. Cryst. Growth 168, 74–92 (1996).

    Article  CAS  Google Scholar 

  35. Saridakis, E. & Chayen, N.E. Systematic improvement of protein crystals by determining the supersolubility curves of phase diagrams. Biophys. J. 84, 1218–1222 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Saridakis, E. & Chayen, N.E. Improving protein crystal quality by decoupling nucleation and growth in vapor diffusion. Protein Sci. 9, 755–757 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nneji, G.A. & Chayen, N.E. A crystallization plate for controlling evaporation in hanging drops. J. Appl. Crystallogr. 37, 502–503 (2004).

    Article  CAS  Google Scholar 

  38. Chayen, N.E. Methods for separating nucleation and growth in protein crystallisation. Prog. Biophys. Mol. Biol. 88, 329–337 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Talreja, S., Kim, D.Y., Mirarefi, A.Y., Zukoski, C.F. & Kenis, P.J.A. Screening and optimization of protein crystallization conditions through gradual evaporation using a novel crystallization platform. J. Appl. Crystallogr. 38, 988–995 (2005).

    Article  CAS  Google Scholar 

  40. D'Arcy, A., Sweeney, A.M. & Haber, A. Practical aspects of using the microbatch method in screening conditions for protein crystallization. Methods 34, 323–328 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Landsberg, M.J., Bond, J., Gee, C.L., Martin, J.L. & Hankamer, B. A method for screening the temperature dependence of three-dimensional crystal formation. Acta Crystallogr. D Biol. Crystallogr. 62, 559–562 (2006).

    Article  PubMed  CAS  Google Scholar 

  42. Przybylska, M. A double cell for controlling nucleation and growth of protein crystals. J. Appl. Crystallogr. 22, 115–118 (1989).

    Article  CAS  Google Scholar 

  43. Saridakis, E.E., Stewart, P.D., Lloyd, L.F. & Blow, D.M. Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2. Acta Crystallogr. D Biol. Crystallogr. 50, 293–297 (1994).

    Article  CAS  PubMed  Google Scholar 

  44. Stura, E.A. & Wilson, I.A. Analytical and production seeding techniques. Methods 1, 38–49 (1990).

    Article  Google Scholar 

  45. Bergfors, T. Seeds to crystals. J. Struct. Biol. 142, 66–76 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Thaller, C. et al. Repeated seeding technique for growing large single crystals of proteins. J. Mol. Biol. 147, 465–469 (1981).

    Article  CAS  PubMed  Google Scholar 

  47. Stura, E.A. & Wilson, I.A. Applications of the streak seeding technique in protein crystallization. J. Cryst. Growth 110, 270–282 (1991).

    Article  CAS  Google Scholar 

  48. Chayen, N.E., Saridakis, E. & Sear, R.P. Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium. Proc. Natl. Acad. Sci. USA 103, 597–601 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Chayen, N.E., Saridakis, E., El Bahar, R. & Nemirovsky, Y. Porous silicon: an effective nucleation-inducing material for protein crystallization. J. Mol. Biol. 312, 591–595 (2001).

    Article  CAS  PubMed  Google Scholar 

  50. Fermani, S., Falini, G., Minnucci, M. & Ripamonti, A. Protein crystallization on polymeric film surfaces. J. Cryst. Growth 224, 327–334 (2001).

    Article  CAS  Google Scholar 

  51. Pechkova, E. & Nicolini, C. Accelerated protein crystal growth by protein thin film template. J. Cryst. Growth 231, 599–602 (2001).

    Article  CAS  Google Scholar 

  52. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

  53. Spangler, B.D. & Westbrook, E.M. Isolation of isoelectrically pure cholera toxin for crystallization. J. Cryst. Growth 110, 220–227 (1991).

    Article  CAS  Google Scholar 

  54. Wilson, W.W. Light scattering as a diagnostic for protein crystal growth—a practical approach. J. Struct. Biol. 142, 56–65 (2003).

    Article  PubMed  CAS  Google Scholar 

  55. Skouri, M., Delsanti, M., Munch, J.P., Lorber, B. & Giege, R. Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions. FEBS Lett. 295, 84–88 (1991).

    Article  CAS  PubMed  Google Scholar 

  56. Mogridge, J. Using light scattering to determine the stoichiometry of protein complexes. Methods Mol. Biol. 261, 113–118 (2004).

    CAS  PubMed  Google Scholar 

  57. Georgalis, Y. & Saenger, W. Light scattering studies on supersaturated protein solutions. Sci. Prog. 82, 271–294 (1999).

    CAS  PubMed  Google Scholar 

  58. Geerlof, A. et al. The impact of protein characterization in structural proteomics. Acta Crystallogr. D Biol. Crystallogr. 62, 1125–1136 (2006).

    Article  PubMed  CAS  Google Scholar 

  59. Ferre-D'Amare, A.R. & Burley, S.K. Use of dynamic light scattering to assess crystallizability of macromolecules and macromolecular assemblies. Structure 2, 357–359 (1994).

    Article  CAS  PubMed  Google Scholar 

  60. Chayen, N.E. et al. Trends and challenges in experimental macromolecular crystallography. Q. Rev. Biophys. 29, 227–278 (1996).

    Article  CAS  PubMed  Google Scholar 

  61. Banci, L. et al. A prokaryotic superoxide dismutase paralog lacking two Cu ligands: from largely unstructured in solution to ordered in the crystal. Proc. Natl. Acad. Sci. USA 102, 7541–7546 (2005).

    Article  CAS  PubMed  Google Scholar 

  62. Banci, L. et al. Fully metallated S134N Cu, Zn-superoxide dismutase displays abnormal mobility and intermolecular contacts in solution. J. Biol. Chem. 280, 35815–35821 (2005).

    Article  CAS  PubMed  Google Scholar 

  63. Liou, Y.C. et al. Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria. Protein Expr. Purif. 19, 148–157 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Mao, H., Gunasekera, A.H. & Fesik, S.W. Expression, refolding, and isotopic labeling of human serum albumin domains for NMR spectroscopy. Protein Expr. Purif. 20, 492–499 (2000).

    Article  CAS  PubMed  Google Scholar 

  65. Jancarik, J., Pufan, R., Hong, C., Kim, S.H. & Kim, R. Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Acta Crystallogr. D Biol. Crystallogr. 60, 1670–1673 (2004).

    Article  PubMed  CAS  Google Scholar 

  66. Liljas, A. et al. Crystal structure of human carbonic anhydrase C. Nat. New Biol. 235, 131–137 (1972).

    Article  CAS  PubMed  Google Scholar 

  67. Arnesano, F. et al. The evolutionarily conserved trimeric structure of CutA1 proteins suggests a role in signal transduction. J. Biol. Chem. 278, 45999–46006 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Lepre, C.A. & Moore, J.M. Microdrop screening: a rapid method to optimize solvent conditions for NMR spectroscopy of proteins. J. Biomol. NMR 12, 493–499 (1998).

    Article  CAS  PubMed  Google Scholar 

  69. Izaac, A., Schall, C.A. & Mueser, T.C. Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searches. Acta Crystallogr. D Biol. Crystallogr. 62, 833–842 (2006).

    Article  PubMed  CAS  Google Scholar 

  70. Collins, B.K., Tomanicek, S.J., Lyamicheva, N., Kaiser, M.W. & Mueser, T.C. A preliminary solubility screen used to improve crystallization trials: crystallization and preliminary x-ray structure determination of Aeropyrum pernix flap endonuclease-1. Acta Crystallogr. D. Biol. Crystallogr. 60, 1674–1678 (2004).

    Article  PubMed  CAS  Google Scholar 

  71. Sazaki, G., Kurihara, K., Nakada, T., Miyashita, S. & Komatsu, H. A novel approach to the solubility measurement of protein crystals by two-beam interferometry. J. Cryst. Growth 169, 355–360 (1996).

    Article  CAS  Google Scholar 

  72. Nakazato, K., Homma, T. & Tomo, T. Rapid solubility measurement of protein crystals as a function of precipitant concentration with micro-dialysis cell and two-beam interferometer. J. Synchrotron Rad. 11, 34–37 (2004).

    Article  CAS  Google Scholar 

  73. Jancarik, J. & Kim, S.H. Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Crystallogr. 24, 409–411 (1991).

    Article  CAS  Google Scholar 

  74. Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y. & McPherson, A. Screening and optimization strategies for macromolecular crystal growth. Acta Crystallogr. D. Biol. Crystallogr. 50, 414–423 (1994).

    Article  CAS  PubMed  Google Scholar 

  75. Tran, T.T., Sorel, I. & Lewit-Bentley, A. Statistical experimental design of protein crystallization screening revisited. Acta Crystallogr. D Biol. Crystallogr. 60, 1562–1568 (2004).

    Article  PubMed  CAS  Google Scholar 

  76. Boistelle, R. & Astier, J.P. Crystallization mechanism in solution. J. Cryst. Growth 90, 14–30 (1988).

    Article  CAS  Google Scholar 

  77. Ng, J.D. et al. The crystallization of biological macromolecules from precipitates: evidence for Ostwald ripening. J. Cryst. Growth 168, 50–62 (1996).

    Article  CAS  Google Scholar 

  78. Vedadi, M. et al. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. USA 103, 15835–15840 (2006).

    Article  CAS  PubMed  Google Scholar 

  79. McPherson, A. et al. The effects of neutral detergents on the crystallization of soluble proteins. J. Cryst. Growth 76, 547–553 (1986).

    Article  CAS  Google Scholar 

  80. Hayes, M.E. et al. Assembly of nucleic acid-lipid nanoparticles from aqueous-organic monophases. Biochim. Biophys. Acta 1758, 429–442 (2006).

    Article  CAS  PubMed  Google Scholar 

  81. Guan, R.J., Wang, M., Liu, X.Q. & Wang, D.C. Optimization of soluble protein crystallization with detergents. J. Cryst. Growth 231, 273–279 (2001).

    Article  CAS  Google Scholar 

  82. Shulgin, I.L. & Ruckenstein, E. Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility. Biophys. Chem. 118, 128–134 (2005).

    Article  CAS  PubMed  Google Scholar 

  83. Tarentino, A.L., Gomez, C.M. & Plummer, T.H. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase. Biochemistry 24, 4665–4671 (1985).

    Article  CAS  PubMed  Google Scholar 

  84. Grueninger-Leitch, F., D'Arcy, A., D'Arcy, B. & Chene, C. Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases. Protein Sci. 5, 2617–2622 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Prota, A.E., Sage, D.R., Stehle, T. & Fingeroth, J.D. The crystal structure of human CD21: Implications for Epstein-Barr virus and C3d binding. Proc. Natl. Acad. Sci. USA 99, 10641–10646 (2002).

    Article  CAS  PubMed  Google Scholar 

  86. Carafoli, F., Chirgadze, D.Y., Blundell, T.L. & Gherardi, E. Crystal structure of the beta-chain of human hepatocyte growth factor-like/macrophage stimulating protein. FEBS J. 272, 5799–5807 (2005).

    Article  CAS  PubMed  Google Scholar 

  87. Nasreen, A., Vogt, M., Kim, H.J., Eichinger, A. & Skerra, A. Solubility engineering and crystallization of human apolipoprotein D. Protein Sci. 15, 190–199 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Roe, S. Protein Purification Applications: a Practical Approach (Oxford University Press, Oxford, UK, New York, 2001).

    Google Scholar 

  89. Liu, J.-W., Boucher, Y., Stokes, H.W. & Ollis, D.L. Improving protein solubility: the use of the Escherichia coli dihydrofolate reductase gene as a fusion reporter. Protein Expr. Purif. 47, 258–263 (2006).

    Article  CAS  PubMed  Google Scholar 

  90. Chernov, A.A. Protein crystals and their growth. J. Struct. Biol. 142, 3–21 (2003).

    Article  CAS  PubMed  Google Scholar 

  91. Chayen, N.E. Comparative studies of protein crystallization by vapour-diffusion and microbatch techniques. Acta Crystallogr. D. Biol. Crystallogr. 54, 8–15 (1998).

    Article  CAS  PubMed  Google Scholar 

  92. McPherson, A. Jr . The growth and preliminary investigation of protein and nucleic acid crystals for x-ray diffraction analysis. Methods Biochem. Anal. 23, 249–345 (1976).

    CAS  PubMed  Google Scholar 

  93. Cudney, R., Patel, S. & McPherson, A. Crystallization of macromolecules in silica gels. Acta Crystallogr. D. Biol. Crystallogr. 50, 479–483 (1994).

    Article  CAS  PubMed  Google Scholar 

  94. Garcia-Ruiz, J.M. Counterdiffusion methods for macromolecular crystallization. Methods Enzymol. 368, 130–154 (2003).

    Article  CAS  PubMed  Google Scholar 

  95. Garcia-Ruiz, J.M., Gonzalez-Ramirez, L.A., Gavira, J.A. & Otalora, F. Granada crystallisation box: a new device for protein crystallisation by counter-diffusion techniques. Acta Crystallogr. D. Biol. Crystallogr. 58, 1638–1642 (2002).

    Article  PubMed  CAS  Google Scholar 

  96. Papanikolau, Y. et al. Ionic strength reducers: an efficient approach to protein purification and crystallization. Application to two Rop variants. Acta Crystallogr. D Biol. Crystallogr. 60, 1334–1337 (2004).

    Article  PubMed  CAS  Google Scholar 

  97. Anand, K., Pal, D. & Hilgenfeld, R. An overview on 2-methyl-2,4-pentanediol in crystallization and in crystals of biological macromolecules. Acta Crystallogr. D Biol. Crystallogr. 58, 1722–1728 (2002).

    Article  PubMed  CAS  Google Scholar 

  98. Berger, B.W., Blamey, C.J., Naik, U.P., Bahnson, B.J. & Lenhoff, A.M. Roles of additives and precipitants in crystallization of calcium- and integrin-binding protein. Cryst. Growth Des. 5, 1499–1507 (2005).

    Article  CAS  Google Scholar 

  99. Wiencek, J.M. New strategies for protein crystal growth. Annu. Rev. Biomed. Eng 1, 505–534 (1999).

    Article  CAS  PubMed  Google Scholar 

  100. Forsythe, E.L., Maxwell, D.L. & Pusey, M. Vapor diffusion, nucleation rates and the reservoir to crystallization volume ratio. Acta Crystallogr. D Biol. Crystallogr. 58, 1601–1605 (2002).

    Article  PubMed  CAS  Google Scholar 

  101. Walter, T.S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D Biol. Crystallogr. 61, 651–657 (2005).

    Article  PubMed  CAS  Google Scholar 

  102. Wolfova, J., Grandori, L., Chatterjee, N., Carey, J. & Smatanova, I.K. Crystallization of the flavoprotein WrbA optimized by using additives and gels. J. Cryst. Growth 284, 502–505 (2005).

    Article  CAS  Google Scholar 

  103. Moreno, A., Theobald-Dietrich, A., Lorber, B., Sauter, C. & Giege, R. Effects of macromolecular impurities and of crystallization method on the quality of eubacterial aspartyl-tRNA synthetase crystals. Acta Crystallogr. D Biol. Crystallogr. 61, 789–792 (2005).

    Article  CAS  PubMed  Google Scholar 

  104. Kinoshita, T., Ataka, M., Warizaya, M., Neya, M. & Fujii, T. Improving quality and harvest period of protein crystals for structure-based drug design: effects of a gel and a magnetic field on bovine adenosine deaminase crystals. Acta Crystallogr. D Biol. Crystallogr. 59, 1333–1335 (2003).

    Article  PubMed  Google Scholar 

  105. Sica, F. et al. Elimination of twinning in crystals of Sulfolobus solfataricus alcohol dehydrogenase holo-enzyme by growth in agarose gels. Acta Crystallogr. D Biol. Crystallogr. 50, 508–511 (1994).

    Article  CAS  PubMed  Google Scholar 

  106. McPherson, A. & Schlichta, P. The use of heterogeneous and epitaxial nucleants to promote the growth of protein crystals. J. Cryst. Growth 90, 47–50 (1988).

    Article  CAS  Google Scholar 

  107. Banci, L. et al. From an inactive prokaryotic SOD homologue to an active protein through site-directed mutagenesis. J. Am. Chem. Soc. 127, 13287–13292 (2005).

    Article  CAS  PubMed  Google Scholar 

  108. Prinz, R. & Weser, U. A naturally occurring Cu-thionein in Saccharomyces cerevisiae . Hoppe Seylers. Z. Physiol Chem. 356, 767–776 (1975).

    Article  CAS  PubMed  Google Scholar 

  109. Luchinat, C. et al. The Cu(I)(7) cluster in yeast copper thionein survives major shortening of the polypeptide backbone as deduced from electronic absorption, circular dichroism, luminescence and (1)H NMR. J. Biol. Inorg. Chem. 8, 353–359 (2003).

    Article  CAS  PubMed  Google Scholar 

  110. Sousa, R. Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization. Acta Crystallogr. D. Biol. Crystallogr. 51, 271–277 (1995).

    Article  CAS  PubMed  Google Scholar 

  111. Farnum, M. & Zukoski, C. Effect of glycerol on the interactions and solubility of bovine pancreatic trypsin inhibitor. Biophys. J. 76, 2716–2726 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Charron, C., Kadri, A., Robert, M.C., Giege, R. & Lorber, B. Crystallization in the presence of glycerol displaces water molecules in the structure of thaumatin. Acta Crystallogr. D. Biol. Crystallogr. 58, 2060–2065 (2002).

    Article  PubMed  CAS  Google Scholar 

  113. Trakhanov, S. & Quiocho, S.A. Influence of divalent cations in protein crystallization. Protein Sci. 4, 1914–1919 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Carbonnaux, C., Ries-Kautt, M. & Ducruix, A. Relative effectiveness of various anions on the solubility of acidic Hypoderma lineatum collagenase at pH 7.2. Protein Sci. 4, 2123–2128 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Vaney, M.C. et al. Structural effects of monovalent anions on polymorphic lysozyme crystals. Acta Crystallogr. D. Biol. Crystallogr. 57, 929–940 (2001).

    Article  CAS  PubMed  Google Scholar 

  116. Ries-Kautt, M.M. & Ducruix, A.F. Relative effectiveness of various ions on the solubility and crystal growth of lysozyme. J. Biol. Chem. 264, 745–748 (1989).

    CAS  PubMed  Google Scholar 

  117. Dauter, Z. & Dauter, M. Entering a new phase. Using solvent halide ions in protein structure determination. Structure 9, R21–R26 (2001).

    Article  CAS  PubMed  Google Scholar 

  118. Dauter, Z., Dauter, M. & Rajashankar, K.R. Novel approach to phasing proteins: derivatization by short cryo-soaking with halides. Acta Crystallogr. D. Biol. Crystallogr. 56, 232–237 (2000).

    Article  CAS  PubMed  Google Scholar 

  119. Dauter, Z. New approaches to high-throughput phasing. Curr. Opin. Struct. Biol. 12, 674–678 (2002).

    Article  CAS  PubMed  Google Scholar 

  120. Lorber, B., Delucas, L.J. & Bishop, J.B. Changes in the physico-chemical properties of the detergent octyl glucoside during membrane protein crystallization using salts as the precipitant. J. Cryst. Growth 110, 103–113 (1991).

    Article  CAS  Google Scholar 

  121. Gebicka, L. & Gebicki, J.L. Kinetic studies on the interaction of ferricytochrome c with anionic surfactants. J. Protein Chem. 18, 165–172 (1999).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank R. Leone for help in collecting some of the crystal images. This work was supported by MIUR (PRIN 2005, Prot. N. 2005039878, Prot. N. 4455) and by the University of Siena PAR2005.

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  1. Dipartimento di Chimica, Università di Siena, Via Aldo Moro 2, Siena, 53100, Italy

    Manuela Benvenuti & Stefano Mangani

  2. CERM, Università di Firenze, Via Luigi Sacconi 6, Firenze, 50019, Italy

    Stefano Mangani

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Correspondence to Stefano Mangani.

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Benvenuti, M., Mangani, S. Crystallization of soluble proteins in vapor diffusion for x-ray crystallography. Nat Protoc 2, 1633–1651 (2007). https://doi.org/10.1038/nprot.2007.198

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