title: AcrB contamination in 2-D crystallization of membrane proteins: lessons from a sodium channel and a putative monovalent cation/proton antiporter.
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.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Christopher A P
authors.initials: CA
authors.lastname: Glover
authors.forename: Vincent L G
authors.initials: VL
authors.lastname: Postis
authors.forename: Kalypso
authors.initials: K
authors.lastname: Charalambous
authors.forename: Svetomir B
authors.initials: SB
authors.lastname: Tzokov
authors.forename: Wesley I
authors.initials: WI
authors.lastname: Booth
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Deacon
authors.forename: B A
authors.initials: BA
authors.lastname: Wallace
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.forename: Per A
authors.initials: PA
authors.lastname: Bullough
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002414:Cations, Monovalent / Q000737:chemistry; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry*; D015222:Sodium Channels / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002414:Cations, Monovalent; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015222:Sodium Channels; C083496:AcrB protein, E coli; C443666:NaChBac protein, bacteria