Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system.
IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 A, and data were collected to 2.9 A resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 A resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 A, beta = 107.9 degrees . An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.
Johnson
Steven
S
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, England.
Roversi
Pietro
P
Espina
Marianela
M
Deane
Janet E
JE
Birket
Susan
S
Picking
William D
WD
Blocker
Ariel
A
Picking
Wendy L
WL
Lea
Susan M
SM
D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D010447:Peptide Hydrolases / Q000378:metabolism; D012763:Shigella flexneri / Q000737:chemistry* / Q000472:pathogenicity
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C095770:IpaD protein, Shigella flexneri; D010447:Peptide Hydrolases