Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the F1 antigen Caf1M-Caf1 chaperone-subunit pre-assembly complex from Yersinia pestis.
The F1 capsular antigen of the plague-causing pathogen Yersinia pestis is assembled from monomeric Caf1 subunits via the Caf1M/Caf1A chaperone/usher system. Y. pestis Caf1M-Caf1 chaperone-subunit complex was purified from the periplasm of Escherichia coli cells overexpressing Caf1M and Caf1 and was crystallized in PEG 4000 solution using hanging-drop vapour diffusion. The crystals diffract to a minimum Bragg spacing of 1.8 A and belong to space group P2(1), with unit-cell parameters a = 36.0, b = 69.2, c = 69.1 A, beta = 93.0 degrees. SeMet-labelled Caf1M-Caf1 complexes were purified and crystallized under the same conditions. The SeMet crystals were identical to the native crystals and diffracted to 1.9 A. Heavy-atom derivative crystals were prepared by soaking in 10 mM K(2)PtCl(4), giving two Pt sites per complex. The experimental electron-density map was obtained by a combination of MAD and MIR methods using both Se- and Pt-derivative crystals.
Zavialov
Anton
A
Department of Molecular Biology, Uppsala Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-753 24 Uppsala, Sweden.
Berglund
Jenny
J
Knight
Stefan D
SD
D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D055672:Static Electricity; D015010:Yersinia pestis / Q000737:chemistry* / Q000235:genetics
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D018832:Molecular Chaperones; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C086725:caf1 protein, Yersinia pestis