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PMID: 19360018 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

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

Nature protocols ·Vol. 4 ·No. 5 ·2009-00-00 ·Pages 619-37

Newby ZE, O'Connell JD, Gruswitz F, Hays FA, Harries WE, Harwood IM, Ho JD, Lee JK, Savage DF, Miercke LJ, Stroud RM

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.

MeSH Terms
Chromatography, Affinity Chromatography, Gel Cloning, Molecular Crystallization/methods Crystallography, X-Ray Detergents Escherichia coli/genetics Membrane Proteins/chemistry,genetics,isolation & purification Protein Structure, Tertiary Solubility
Chemicals
Detergents Membrane Proteins
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Newby Zachary E R
Department of Biochemistry and Biophysics, University of California in San Francisco, 600 16th Street, San Francisco, California 94158-2517, USA.
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
Stroud Robert M
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Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2009.27
Published
2009-00-00
Pages
619-37
Language
English
Region
England
NLM ID
101284307
PMCID
PMC4075773
Subset
IM
Grants
NIGMS NIH HHS · R01 GM024485 · United States
NIGMS NIH HHS · P50 GM073210-04 · United States
NIGMS NIH HHS · R01 GM24485 · United States
NIGMS NIH HHS · P50 GM073210-05 · United States
NIGMS NIH HHS · U54 GM074929 · United States
NIGMS NIH HHS · P50 GM073210-02 · United States
NIGMS NIH HHS · P50 GM074929-01 · United States
NIGMS NIH HHS · P50 GM073210-03 · United States
NIGMS NIH HHS · P50 GM073210-01 · United States
NIGMS NIH HHS · P50 GM073210 · United States
External Links
PubMed source DOI: 10.1038/nprot.2009.27 PMC Full Text View journal details
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