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Acta Crystallogr F Struct Biol Commun. 2017 Jun 1;73(Pt 6):321-327. doi: 10.1107/S2053230X17006963. Epub 2017 May 25.

Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG.

Author information

1
Laboratory of Structural Biology, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad 121 001, India.
2
Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.

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.

KEYWORDS:

Lactobacillus rhamnosus GG; SpaE; adhesion; basal pilin; host–microbe interaction; probiotics; sortase-dependent pili

PMID:
28580919
DOI:
10.1107/S2053230X17006963
[Indexed for MEDLINE]

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