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Title: Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved

Abstract

A method is presented that allowed the diffraction limit of crystals of the secreted chorismate mutase from M. tuberculosis to be improved from approximately 3.5 to 1.3 Å. To obtain large well diffracting crystals, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min. Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ{sub γ} subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000 ▶), Protein Sci.9, 755–757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 Å resolution. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 Å, βmore » = 104.5°. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.« less

Authors:
 [1];  [2];  [1];  [3];  [1];  [2]; ;  [3];  [4]
  1. Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg (Sweden)
  2. (Norway)
  3. Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich (Switzerland)
  4. (Sweden)
Publication Date:
OSTI Identifier:
22356324
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 5; Other Information: PMCID: PMC2219981; PMID: 16682771; PUBLISHER-ID: pu5123; OAI: oai:pubmedcentral.nih.gov:2219981; Copyright (c) International Union of Crystallography 2006; This is an open-access article distributed under the terms described at http://journals.iucr.org/services/termsofuse.html.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CONVERSION; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; DIMERS; NUCLEATION; OPTIMIZATION; PROTEINS; RESOLUTION; SPACE GROUPS

Citation Formats

Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Dey, Raja, Sasso, Severin, Ökvist, Mats, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Ramakrishnan, Chandra, Kast, Peter, and Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg. Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106012036.
Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Dey, Raja, Sasso, Severin, Ökvist, Mats, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Ramakrishnan, Chandra, Kast, Peter, & Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg. Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved. United Kingdom. doi:10.1107/S1744309106012036.
Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Dey, Raja, Sasso, Severin, Ökvist, Mats, Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Ramakrishnan, Chandra, Kast, Peter, and Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg. Mon . "Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved". United Kingdom. doi:10.1107/S1744309106012036.
@article{osti_22356324,
title = {Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved},
author = {Krengel, Ute, E-mail: ute.krengel@kjemi.uio.no and Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo and Dey, Raja and Sasso, Severin and Ökvist, Mats and Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo and Ramakrishnan, Chandra and Kast, Peter and Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg},
abstractNote = {A method is presented that allowed the diffraction limit of crystals of the secreted chorismate mutase from M. tuberculosis to be improved from approximately 3.5 to 1.3 Å. To obtain large well diffracting crystals, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min. Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ{sub γ} subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000 ▶), Protein Sci.9, 755–757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 Å resolution. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 Å, β = 104.5°. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.},
doi = {10.1107/S1744309106012036},
journal = {Acta Crystallographica. Section F},
number = Pt 5,
volume = 62,
place = {United Kingdom},
year = {Mon May 01 00:00:00 EDT 2006},
month = {Mon May 01 00:00:00 EDT 2006}
}