Abstract
Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21–25-Å resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component.
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Change history
18 May 2009
In the version of this article initially published, the incorrect accession number for EM map deposition was provided. The correct accession number for data deposited in EmDep is EMD-1617. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank B. Lin (Brookhaven) for assistance with STEM sample preparation and P. Roversi (Oxford) for the script to run the program for statistical assessment of docking of atomic structures to EM maps. We are indebted to N. Strynadka and co-workers (British Columbia) for the EscJ ring coordinates and to D. DeRosier (Brandeis) and K. Namba (GSFBS, Osaka) for advice and encouragement at all key stages of this work. They, along with F. Booy (Bristol), S. Daniell (Bristol), A. Veenendaal (Utrecht) and W. Steffen (MHH, Hanover) are also thanked for critical comments on the manuscript. J.L.H. was funded by UK Medical Research Council project grant G0401595 to A.J.B. and Deutsche Forschungsgemeinschaft grant BR 849/29-1 to B. Brenner. A.J.B. was supported by the Guy G. F. Newton Senior Research Fellowship. S.J. was funded by UK Medical Research Council project grant G0400389 to S.M.L. J.S.W.'s laboratory is supported by the US National Institutes of Health and Department of Energy. P.C.A.d.F. and E.P.M. received funding from Cancer Research UK.
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A.J.B. and J.L.H. designed the research; D.S. and A.J.B. purified the needle complexes; J.L.H. acquired the negative stain images; M.S. acquired the STEM images; J.S.W. adapted the STEM image analysis programme; J.L.H., A.H. and A.J.B. analyzed data; P.C.A.d.F. and E.P.M. provided advice on image analysis; S.J., J.L.H. and S.M.L. performed the EscJ docking; J.L.H. and A.J.B. wrote the paper.
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Hodgkinson, J., Horsley, A., Stabat, D. et al. Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nat Struct Mol Biol 16, 477–485 (2009). https://doi.org/10.1038/nsmb.1599
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DOI: https://doi.org/10.1038/nsmb.1599
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