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Updated: Sep 27, 2026
From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
Published on: July 4, 2016
Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the F1 antigen Caf1M-Caf1
Anton Zavialov1, Jenny Berglund, Stefan D Knight
1Department of Molecular Biology, Uppsala Biomedical Center, Swedish University of Agricultural Sciences, Box 590, SE-753 24 Uppsala, Sweden.
Abstract:
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.
