Determination of Soluble and Membrane Protein Structures by X-Ray Crystallography

  • Raquel L. Lieberman
  • Mary E. Peek
  • J. Derrick Watkins
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 955)

Abstract

X-ray crystallography is a technique used to determine the atomic-detail structure of a biological macromolecule. The method relies on the ability to generate a three-dimensional crystal of a highly purified protein or nucleic acid for diffraction by X-rays. The extent of scattering of X-rays by the crystal determines the accuracy of the resulting structural model. Unlike electrons, X-rays cannot be refocused after they have been scattered by their target. Thus, calculations are needed to reconstruct the image of the macromolecule that builds the crystal lattice. Tremendous advances over the past 60 years in recombinant expression and purification, crystal growth methods and equipment, X-ray sources, computer processing power, programs, and graphics have taken X-ray crystallography from a highly specialized field to one increasingly accessible to researchers in the biomedical sciences. In this chapter, we review the major concepts of macromolecular X-ray crystallography, focusing mainly on techniques for crystallizing soluble and membrane proteins, and provide a protocol for the crystallization of lysozyme as a model for the crystallization of other proteins.

Key words

Crystals Vapor diffusion Fourier transform Model building Refinement Protein data bank Membrane protein Lysozyme 
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Notes

Acknowledgments

This work was supported by grants to RLL from NSF (CAREER award 0845445) and the American Federation for Aging Research. Diffraction data were collected at the Southeast Regional Collaborative Access Team (Ser-CAT) beamline 22-BM at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. We acknowledge the feedback on lysozyme crystallization from undergraduate students enrolled in CHEM 4582, 4681, as well as visitors from Westlake High School. This manuscript is dedicated to the memory of biology teacher Ayesha Johnson.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Raquel L. Lieberman
    • 1
  • Mary E. Peek
    • 1
  • J. Derrick Watkins
    • 1
  1. 1.School of Chemistry and BiochemistryGeorgia Institute of TechnologyAtlantaUSA

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