Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system.

Journal article

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.

Authors: Roversi, P; Johnson, S; Field, T; Deane, J; Galyov, E

• Publication status: Published
• Journal: Acta crystallographica. Section F, Structural biology and crystallization communications
• Volume: 62
• Issue: Pt 9
• Pages: 861-864
• Publication date: 2006-09-01
• DOI: 10.1107/s1744309106027035
• EISSN: 1744-3091
• ISSN: 1744-3091
• Language: English
• Keywords: Gene Expression Regulation, Bacterial; Virulence Factors; Peptide Fragments; Burkholderia pseudomallei; Crystallization; Crystallography, X-Ray; Bacterial Proteins