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    Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

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    Picking_2006.pdf (381.3Kb)
    Issue Date
    2006-08-11
    Author
    Johnson, Steven
    Roversi, Pietro
    Espina, Marianela
    Deane, Janet E.
    Birket, Susan
    Picking, William D.
    Picking, Wendy Lynn
    Lea, Susan M.
    Publisher
    International Union of Crystallography
    Type
    Article
    Article Version
    Scholarly/refereed, publisher version
    Rights
    Copyright © International Union of Crystallography 2006. This is an open-access article distributed under the terms described at http://journals.iucr.org/services/termsofuse.html.
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    Abstract
    aD, 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 P212121, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å 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 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. 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.
    URI
    http://hdl.handle.net/1808/21169
    DOI
    https://doi.org/10.1107/S1744309106027047
    Collections
    • Pharmaceutical Chemistry Scholarly Works [327]
    Citation
    Johnson, S., Roversi, P., Espina, M., Deane, J. E., Birket, S., Picking, W. D., … Lea, S. M. (2006). Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 62(Pt 9), 865–868. http://doi.org/10.1107/S1744309106027047

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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