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

• Published in: Nature protocols Vol. 4; no. 5; pp. 619 - 637
• Main Authors: Stroud, Robert M, Newby, Zachary E R, O'Connell, Joseph D, Gruswitz, Franz, Hays, Franklin A, Harries, William E C, Harwood, Ian M, Ho, Joseph D, Lee, John K, Savage, David F, Miercke, Larry J W
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.01.2009; Nature Publishing Group
• Subjects: Analytical Chemistry; Biological Techniques; Biomedical and Life Sciences; Chromatography, Affinity; Chromatography, Gel; Cloning, Molecular; Computational Biology/Bioinformatics; Crystallization; Crystallization - methods; Crystallography; Crystallography, X-Ray; Crystals; Deoxyribonucleic acid; Detergents; DNA; Escherichia coli - genetics; Ions; Life Sciences; Membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membranes; Methods; Microarrays; Organic Chemistry; Protein research; Protein Structure, Tertiary; Proteins; Protocol; Solubility; Structure; X-ray crystallography; X-ray diffraction; Yeast; United States; United States > US; San Francisco California; California
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2009.27

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.