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J Struct Biol. 2015 Feb;189(2):123-34. doi: 10.1016/j.jsb.2014.11.008. Epub 2014 Dec 3.

Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization.

Author information

1
New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA.
2
Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA.
3
Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
4
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
5
New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA; Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
6
New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA; Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA. Electronic address: Iban.Ubarretxena@mssm.edu.

Abstract

Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.

KEYWORDS:

96-well format; Electron cryomicroscopy; Electron crystallography; High-throughput screening; Membrane protein; Membrane protein reconstitution; Two-dimensional (2D) crystal

PMID:
25478971
PMCID:
PMC4419781
DOI:
10.1016/j.jsb.2014.11.008
[Indexed for MEDLINE]
Free PMC Article

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