Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system.
A construct consisting of residues 10-310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 A, a calculated Matthews coefficient of 2.4 A(3) Da(-1) (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P2(1)2(1)2, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 A, and a calculated Matthews coefficient of 2.3 A(3) Da(-1) (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P2(1)2(1)2 form). The native crystals of both forms give diffraction data to 2.7 A resolution, while the SeMet-labelled P2(1)2(1)2 crystals diffract to 3.3 A resolution. A K(2)PtCl(4) derivative of the P2(1)2(1)2 form was also obtained and data were collected to 2.7 A with radiation of wavelength lambda = 0.933 A. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.
Roversi
Pietro
P
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England.
Johnson
Steven
S
Field
Terry
T
Deane
Janet E
JE
Galyov
Edouard E
EE
Lea
Susan M
SM
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016957:Burkholderia pseudomallei / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015964:Gene Expression Regulation, Bacterial; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D037521:Virulence Factors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D010446:Peptide Fragments; D037521:Virulence Factors