Open AccessCrystallization and preliminary X‐ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy‐terminal domain of TonB
Author(s) -
Shultis David D.,
Purdy Michael D.,
Banchs Christian N.,
Wiener Michael C.
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106018240
Subject(s) - escherichia coli , cobalamin , crystallization , crystallography , bacterial outer membrane , transporter , chemistry , membrane , biophysics , biochemistry , biology , gene , vitamin b12 , organic chemistry
The energy‐dependent uptake of organometallic compounds and other micronutrients across the outer membranes of Gram‐negative bacteria is carried out by outer membrane active‐transport proteins that utilize the proton‐motive force of the inner membrane via coupling to the TonB protein. The Escherichia coli outer membrane cobalamin transporter BtuB and a carboxy‐terminal domain of the TonB protein, residues 147–239 of the wild‐type protein, were expressed and purified individually. A complex of BtuB and TonB 147–239 was formed in the presence of the substrate cyanocobalamin (CN‐Cbl; vitamin B 12 ) and calcium and was crystallized. BtuB was purified in the detergent LDAO ( n ‐dodecyl‐ N , N ‐dimethylamine‐ N ‐oxide) and the complex was formed in a detergent mixture of LDAO and C 8 E 4 (tetraethylene glycol monooctylether). Crystals were obtained by sitting‐drop vapor diffusion, with the reservoir containing 30%( v / v ) polyethylene glycol (PEG 300) and 100 m M sodium acetate pH 5.2. The crystals belong to space group P 2 1 2 1 2 1 (unit‐cell parameters a = 74.3, b = 82.4, c = 122.6 Å). The asymmetric unit consists of a single BtuB–TonB complex. Data sets have been collected to 2.1 Å resolution at a synchrotron beamline (APS SER‐CAT 22‐ID).