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

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Protocol publication

[…] Crystal forms 1, 2, 3, 4A and 5 were cryoprotected in reservoir solution containing 20%(v/v) glycerol, 20%(v/v) PEG 400, 5%(v/v) PEG 400, 25%(v/v) ethylene glycol and 5%(v/v) PEG 400, respectively, and flash-cryocooled in liquid nitrogen prior to data collection. Crystal form 4B was mounted and flash-cryocooled directly in the cryostream.Diffraction data were recorded at 100 K (Table 1). Data were indexed and integrated in MOSFLM (Leslie, 1992) and scaled with SCALA (Evans, 1997) within the CCP4 program suite (Collaborative Computational Project, Number 4, 1994). A fluorescence scan on a SeMet crystal (CF-4B) across the Se edge yielded values of = −8.0 and = 5.2 using the program CHOOCH (Evans & Pettifer, 2001). […]

Pipeline specifications

Software tools iMosflm, CCP4
Applications Small-angle scattering, Protein structure analysis
Organisms Shigella flexneri
Chemicals Selenomethionine