Mishra, Arjun K. and Megta, Abhin Kumar and Palva, Airi and von Ossowski, Ingemar and Krishnan, Vengadesan (2017) Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG. Acta Crystallographica Section F Structural Biology Communications, 73 (6). pp. 321-327. ISSN 2053-230X

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Abstract

SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Å were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Å resolution.

Item Type:	Article
Subjects:	Biomedical Science Biochemical and Biophysical Sciences
Depositing User:	Dr Vengadesan Krishnan
Date Deposited:	12 Sep 2017 09:04
Last Modified:	20 Feb 2018 11:33
URI:	http://rcb.sciencecentral.in/id/eprint/35

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