title: Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.
abstract: TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.
authors.affiliation: Biozentrum, University of Basel, Klingelbergstr. 70, 4056, Basel, Switzerland.
authors.forename: Fabian
authors.initials: F
authors.lastname: Gruss
authors.affiliation: Biozentrum, University of Basel, Klingelbergstr. 70, 4056, Basel, Switzerland. sebastian.hiller@unibas.ch.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hiller
authors.affiliation: Biozentrum, University of Basel, Klingelbergstr. 70, 4056, Basel, Switzerland. timm.maier@unibas.ch.
authors.forename: Timm
authors.initials: T
authors.lastname: Maier
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D003460:Crystallization / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008823:Micelles; D010957:Plasmids / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008823:Micelles
keywords: Autotransporter; Bacterial outer membrane; Bicelle crystallization; Membrane protein purification; Omp85; Outer membrane protein; TamA; X-ray crystallography; β-Barrel