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Title: Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB

Abstract

Crystals of a complex of the E. coli proteins BtuB (outer membrane cobalamin transporter) and TonB (carboxy-terminal domain) diffracting to 2.1 Å resolution have been obtained. 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{sup 147–239} was formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B{sub 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{sub 8}E{sub 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 mM sodium acetate pH 5.2. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 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 havemore » been collected to 2.1 Å resolution at a synchrotron beamline (APS SER-CAT 22-ID)« less

Authors:
;  [1];  [2]
  1. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908 (United States)
  2. Interdisciplinary Graduate Program in Biophysics, University of Virginia, Charlottesville, Virginia 22908 (United States)
Publication Date:
OSTI Identifier:
22360210
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section F
Additional Journal Information:
Journal Volume: 62; Journal Issue: Pt 7; Other Information: PMCID: PMC2242962; PMID: 16820681; PUBLISHER-ID: pu5141; OAI: oai:pubmedcentral.nih.gov:2242962; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 1744-3091
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ACETATES; CALCIUM; COUPLING; CRYSTALLIZATION; CRYSTALS; DIFFUSION; ESCHERICHIA COLI; MEMBRANES; MIXTURES; OXIDES; RESOLUTION; SODIUM; SPACE GROUPS; SUBSTRATES

Citation Formats

Shultis, David D., Purdy, Michael D., Banchs, Christian N., Wiener, Michael C., E-mail: mwiener@virginia.edu, and Interdisciplinary Graduate Program in Biophysics, University of Virginia, Charlottesville, Virginia 22908. Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106018240.
Shultis, David D., Purdy, Michael D., Banchs, Christian N., Wiener, Michael C., E-mail: mwiener@virginia.edu, & Interdisciplinary Graduate Program in Biophysics, University of Virginia, Charlottesville, Virginia 22908. Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB. United Kingdom. https://doi.org/10.1107/S1744309106018240
Shultis, David D., Purdy, Michael D., Banchs, Christian N., Wiener, Michael C., E-mail: mwiener@virginia.edu, and Interdisciplinary Graduate Program in Biophysics, University of Virginia, Charlottesville, Virginia 22908. 2006. "Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB". United Kingdom. https://doi.org/10.1107/S1744309106018240.
@article{osti_22360210,
title = {Crystallization 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 = {Shultis, David D. and Purdy, Michael D. and Banchs, Christian N. and Wiener, Michael C., E-mail: mwiener@virginia.edu and Interdisciplinary Graduate Program in Biophysics, University of Virginia, Charlottesville, Virginia 22908},
abstractNote = {Crystals of a complex of the E. coli proteins BtuB (outer membrane cobalamin transporter) and TonB (carboxy-terminal domain) diffracting to 2.1 Å resolution have been obtained. 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{sup 147–239} was formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B{sub 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{sub 8}E{sub 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 mM sodium acetate pH 5.2. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 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)},
doi = {10.1107/S1744309106018240},
url = {https://www.osti.gov/biblio/22360210}, journal = {Acta Crystallographica. Section F},
issn = {1744-3091},
number = Pt 7,
volume = 62,
place = {United Kingdom},
year = {Sat Jul 01 00:00:00 EDT 2006},
month = {Sat Jul 01 00:00:00 EDT 2006}
}