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Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system
Johnson, Steven Roversi, Pietro Espina, Marianela Deane, Janet E. Birket, Susan Picking, William D. Picking, Wendy Lynn Lea, Susan M.
Johnson, Steven
Roversi, Pietro
Espina, Marianela
Deane, Janet E.
Birket, Susan
Picking, William D.
Picking, Wendy Lynn
Lea, Susan M.
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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.
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Date
2006-08-11
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International Union of Crystallography
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Keywords
IpaD, Type III secretion, Shigella flexneri
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