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AcrB contamination in 2-D crystallization of membrane proteins: lessons from a sodium channel and a putative monovalent cation/proton antiporter.

Authors :
Glover CA
Postis VL
Charalambous K
Tzokov SB
Booth WI
Deacon SE
Wallace BA
Baldwin SA
Bullough PA
Source :
Journal of structural biology [J Struct Biol] 2011 Dec; Vol. 176 (3), pp. 419-24. Date of Electronic Publication: 2011 Sep 20.
Publication Year :
2011

Abstract

Contamination with the multidrug transporter AcrB represents a potential pitfall in the structural analysis of recombinant membrane proteins expressed in Escherichia coli, especially when high-throughput approaches are adopted. This can be a particular problem in two-dimensional (2-D) crystallization for electron cryomicroscopy since individual crystals are too small for compositional analysis. Using a broad 'sparse matrix' of buffer conditions typically used in 2-D crystallization, we have identified at least eight unique crystal forms of AcrB. Reference to images and projection maps of these different forms can greatly facilitate the early identification of false leads in 2-D crystallization trials of other membrane proteins of interest. We illustrate the usefulness of such data by highlighting two studies of membrane proteins in our laboratories. We show in one case (a bacterial sodium channel, NaChBac) how early crystallization 'hits' could be attributed to contaminating AcrB by comparison against our AcrB crystal image database. In a second case, involving a member of the monovalent cation/proton antiporter-1 family (MPSIL0171), a comparison with the observed AcrB crystal forms allowed easy identification of reconstituted AcrB particles, greatly facilitating the eventual purification and crystallization of the correct protein in pure form as ordered helical arrays. Our database of AcrB crystal images will be of general use in assisting future 2-D crystallization studies of other membrane proteins.<br /> (Copyright © 2011 Elsevier Inc. All rights reserved.)

Details

Language :
English
ISSN :
1095-8657
Volume :
176
Issue :
3
Database :
MEDLINE
Journal :
Journal of structural biology
Publication Type :
Academic Journal
Accession number :
21964467
Full Text :
https://doi.org/10.1016/j.jsb.2011.09.005