Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle.

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
Janet E DeaneAriel Blocker

Abstract

A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiH(CDelta5) and diffraction data were collected to 1.9 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 A, beta = 96.5 degrees. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 A3 Da(-1) for two molecules per asymmetric unit, corresponding to a solvent content of 33%.

References

Apr 28, 1968·Journal of Molecular Biology·B W Matthews
Feb 7, 2003·The Journal of Biological Chemistry·Frank S CordesSusan M Lea
Mar 13, 2003·Proceedings of the National Academy of Sciences of the United States of America·Ariel BlockerShin-Ichi Aizawa
Oct 27, 2004·Acta Crystallographica. Section D, Biological Crystallography·Fadel A SamateyKeiichi Namba
Oct 18, 2005·The Journal of Biological Chemistry·Roma KenjaleAriel Blocker
Nov 3, 2005·Current Opinion in Structural Biology·Steven JohnsonSusan M Lea

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Citations

Aug 5, 2006·Proceedings of the National Academy of Sciences of the United States of America·Janet E DeaneSusan M Lea
May 7, 2008·Proceedings of the National Academy of Sciences of the United States of America·Ariel J BlockerSusan M Lea
Jul 8, 2011·The Journal of Biological Chemistry·Melanie A McDowellSusan M Lea
Sep 2, 2006·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Pietro RoversiSusan M Lea
Sep 2, 2006·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Steven JohnsonSusan M Lea
Jan 24, 2015·Acta Crystallographica. Section F, Structural Biology Communications·Ha-Neul KimMin-Duk Seo
Mar 20, 2014·Infection and Immunity·Danielle L JessenMatthew L Nilles
Jun 15, 2010·Nature Structural & Molecular Biology·Omer PoyrazMichael Kolbe
Nov 11, 2008·Journal of Bacteriology·Andreas K J VeenendaalAriel J Blocker
Mar 22, 2016·Microbiology Spectrum·Erin R Green, Joan Mecsas

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Methods Mentioned

BETA
gel-filtration

Software Mentioned

MOSFLM
SCALA
autoSHARP
CCP

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