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

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Abstract

IpaD, 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 P2(1)2(1)2(1), with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 A, and data were collected to 2.9 A 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 A resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 A, beta = 107.9 degrees . 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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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(9), 865–868. https://doi.org/10.1107/S1744309106027047

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