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Macromolecular Crystallography Protocols pp 1–18Cite as

Cryocrystallography of Macromolecules

Practice and Optimization

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 364))

Abstract

Techniques for flash-cooling protein crystals to around 100K (−173°C) for data collection have developed enormously in the last decade, to the extent that cryocrystallography is now standard practice. The main advantage of these methods is the vastly reduced rate of radiation damage to protein crystals in the X-ray beam at cryogenic temperatures over room temperature, extending their lifetimes so that complete datasets can be collected from a single crystal. The practical application of the techniques has become somewhat anecdotal and rather fixed within individual laboratories.

This chapter gives step-by-step guidelines for flash-cooling crystals and some of the rationale for the recommended procedures. Optimization of the entire cryoprotocol can give substantial improvements to both the resolution and quality of the data, often resulting in more straightforward structure solution and subsequent model refinement. Attention to seemingly insignificant details can have a real impact on the usefulness of the final dataset, and are thus worth addressing.

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References

  1. Rodgers, D. W. (1997) Practical cryocrystallography. In: Methods in Enzymology, vol. 276, (Carter, C. W., Jr., and Sweet, R. M., eds.), Academic Press, San Diego, CA, pp. 183–202.

    Google Scholar 

  2. Garman, E. and Schneider, T. (1997) Macromolecular cryocrystallography. J. Appl. Cryst. 27, 211–237.

    Article  Google Scholar 

  3. Parkin, S. and Hope, H. (1998) Macromolecular cryocrystallography: cooling, mounting, storage and transportation of crystals. J. Appl. Cryst. 31, 945–953.

    Article  CAS  Google Scholar 

  4. Garman, E. (1999) Cool data: quality and quantity. Acta Cryst. D55, 1641–1653.

    CAS  Google Scholar 

  5. Hope, H. (2001) Introduction to cryocrystallography. In: International Tables for Crystallography: Volume F, Crystallography of Biological Macromolecules, (Rossmann, M., Arnold, G., and Dordrecht, E., eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 197–201.

    Google Scholar 

  6. Rodgers, D. W. (2001) Cryocrystallography techniques and devices. In: International Tables for Crystallography: Volume F, Crystallography of Biological Macromolecules, (Rossmann, M. G., and Arnold, E. eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 202–208.

    Google Scholar 

  7. Henderson, R. (1990) Cryo-protection of protein crystals against radiation damage in electron and X-ray diffraction. Proc. R. Soc. Lond. B 241, 6–8.

    Article  CAS  Google Scholar 

  8. Weik, M., Ravelli, R. G. B., Kryger, G., et al. (2000) Specific chemical and structural damage to proteins produced by synchrotron radiation. PNAS 97, 623–628.

    Article  PubMed  CAS  Google Scholar 

  9. Burmeister, W. P. (2000) Structural changes in a cryo-cooled protein crystal owing to radiation damage. Acta Cryst. D56, 328–341.

    Google Scholar 

  10. Ravelli, R. G. B. and McSweeney, S. (2000) The ‘fingerprint’ that X-rays can leave on structures. Structure 8, 315–328.

    Article  PubMed  CAS  Google Scholar 

  11. Garman, E. (2003) ‘Cool’ crystals: cryocrystallography and radiation damage. Curr. Opin. Struct. Biol. 13, 545–551.

    Article  PubMed  CAS  Google Scholar 

  12. Teng T-Y. (1990) Mounting of crystals for macromolecular crystallography in a free-standing thin-film. J. Appl. Cryst. 23, 387–391.

    Article  CAS  Google Scholar 

  13. Garman, E. and Doublié, S. (2003) Cryocooling of Macromolecular Crystals: Optimisation methods. Methods in Enzymology, vol. 368, (Carter, C. W., Jr. and Sweet, R. M., eds.), Academic Press, San Diego, CA, pp. 190–217.

    Google Scholar 

  14. Jancarik, J. and Kim, S.-H. (1991) Sparse matrix smpling: a screening method for crystallisation of proteins. J. Appl. Cryst. 24, 409–411.

    Article  CAS  Google Scholar 

  15. Garman, E. F. and Mitchell, E. M. (1996) Glycerol concentrations required for cryoprotection of 50 typical protein crystallisation solutions. J. Appl. Cryst. 29, 584–587.

    Article  CAS  Google Scholar 

  16. McFerrin, M. and Snell, E. (2002) The development and application of a method to quantify the quality of cryoprotectant solutions using standard area-detector X-ray images. J. Appl. Cryst. 35, 538–545.

    Article  CAS  Google Scholar 

  17. Rubinson, K. A., Ladner, J. E., Tordova, M., and Gilliland, G. L. (2000) Cryosalts: suppression of ice formation in macromolecular crystallography. Acta Cryst. D56, 996–1001.

    CAS  Google Scholar 

  18. Wierenga, R. K., Zeelan, J. P., and Noble, M. E. (1992) Crystal transfer experiments carried out with crystals of triosephosphate isomerase (TIM) J. Cryst. Growth 122, 231–234.

    Article  CAS  Google Scholar 

  19. Mitchell, E. M. and Garman, E. F. (1994) Flash freezing of protein crystals: investigation of mosaic spread diffraction limit with cryoprotectant concentration. J. Appl. Cryst 27, 1070–1074.

    Article  CAS  Google Scholar 

  20. Garman, E. F. and Owen, R. L. (2006) Cryocooling and radiation damage in macromolecular crystallography. Acta Cryst. D62, 32–47.

    CAS  Google Scholar 

  21. Weik, M., Kryger, G., Schreurs, A. M. M., et al. (2001) Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures. Acta Cryst. D57, 566–573.

    CAS  Google Scholar 

  22. Kriminski, S., Kazmierczak, M., and Thorne, R. E. (2003) Heat transfer from protein crystals: implication for flash-cooling and X-ray beam heating. Acta Cryst. D59, 697–708.

    CAS  Google Scholar 

  23. Skrzypczak-Jankun, E., Bianchet, M. A., Amzel, L. M., and Funk, M.O., Jr. (1996) Flash-freezing causes a stress-induced modulation in a crystal structure of soybean lipoxygenase L3. Acta Cryst. D52, 959–965.

    CAS  Google Scholar 

  24. Garman, E. F. (1999) Crystallisation for cryo-data collection. In: Protein Crystallisation: Techniques, Strategies and Tips, (Bergfors, T. M., ed.), International University Line, La Jolla, CA, pp. 183–195.

    Google Scholar 

  25. Garman, E. F. (2001) Macromolecular cryo-crystallography. In: Methods in Macromolecular Crystallography (Turk, D. and Johnson, L., eds.), IOS Press, Amsterdam, The Netherlands, pp. 15–25.

    Google Scholar 

  26. Yeh, J. I. and Hol, W. G. J. (1998) A flash-annealing technique to improve diffraction limits and lower mosaicity in crystals of glycerol kinase. Acta Cryst. D54, 479–480.

    CAS  Google Scholar 

  27. Hanson, B. L., Schall, C. A., and Bunick, G. J. (2003) New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium. J. Struct. Biol. 142, 77–87.

    Article  PubMed  Google Scholar 

  28. Kriminski, S., Caylor, C. L., Nonato, M. C., Finkelstein, K. D., and Thorne, R. E. (2003) Flash-cooling and annealing of protein crystals. Acta Cryst. D58, 459–471.

    Google Scholar 

  29. Juers, D. H. and Mathews, B. W. (2004) The role of solvent transport in cryoannealing of macromolecular crystals. Acta Cryst. D60, 412–421.

    CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Biochemistry, Laboratory of Molecular Biophysics, University of Oxford, Oxford, UK

    Elspeth Garman

  2. Department of Biochemistry, University of Oxford, Oxford, UK

    Robin L. Owen

Authors
  1. Elspeth Garman
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  2. Robin L. Owen
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Editor information

Editors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of Vermont, Burlingon, VT

    Sylvie Doublié

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© 2007 Humana Press Inc.

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Garman, E., Owen, R.L. (2007). Cryocrystallography of Macromolecules. In: Doublié, S. (eds) Macromolecular Crystallography Protocols. Methods in Molecular Biology™, vol 364. Humana Press. https://doi.org/10.1385/1-59745-266-1:1

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  • DOI: https://doi.org/10.1385/1-59745-266-1:1

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-902-4

  • Online ISBN: 978-1-59745-266-3

  • eBook Packages: Springer Protocols

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