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A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Nature Protocols volume 4, pages 619–637 (2009)Cite this article

Abstract

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.

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Figure 1: Workflow for generating membrane protein crystals.
Figure 2: Detergent solubilization of membrane proteins.
Figure 3: Size-exclusion chromatography.
Figure 4: Example of grid screens.
Figure 5: Scoring membrane protein crystal trials.
Figure 6: Flowchart for crystal optimization.

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Acknowledgements

Research was supported by NIH grant RO1 GM24485 to R.M.S., the NIH Roadmap center grant P50 GM073210 and the Specialized Center grant of the Protein Structure Initiative U54 GM074929-01. We thank Rebecca Robbins in the Stroud laboratory and Ryan R Atkinson for helpful discussions and help in preparing the manuscript. We also thank the reviewers and editors for their helpful suggestions.

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  1. David F Savage

    Present address: Present address: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.,

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California in San Francisco, 600 16th Street, San Francisco, 94158-2517, California, USA

    Zachary E R Newby, Joseph D O'Connell III, Franz Gruswitz, Franklin A Hays, William E C Harries, Ian M Harwood, Joseph D Ho, John K Lee, David F Savage, Larry J W Miercke & Robert M Stroud

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Correspondence to Robert M Stroud.

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Newby, Z., O'Connell, J., Gruswitz, F. et al. A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat Protoc 4, 619–637 (2009). https://doi.org/10.1038/nprot.2009.27

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