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

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Mishra , A K , Megta , A K , Palva , A , von Ossowski , I & Krishnan , V 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 , vol. 73 , pp. 321-327 . https://doi.org/10.1107/S2053230X17006963

Title: Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG
Author: Mishra, Arjun K.; Megta, Abhin Kumar; Palva, Airi; von Ossowski, Ingemar; Krishnan, Vengadesan
Contributor: University of Helsinki, Departments of Faculty of Veterinary Medicine
University of Helsinki, Departments of Faculty of Veterinary Medicine
Date: 2017-06
Language: eng
Number of pages: 7
Belongs to series: Acta crystallographica section f-Structural biology communications
ISSN: 2053-230X
URI: http://hdl.handle.net/10138/214797
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 angstrom 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 angstrom. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 angstrom resolution.
Subject: adhesion
probiotics
basal pilin
host-microbe interaction
sortase-dependent pili
Lactobacillus rhamnosus GG
SpaE
GRAM-POSITIVE BACTERIA
BACKBONE-PILIN SUBUNIT
CRYSTALLOGRAPHIC ANALYSIS
FUNCTIONAL-ANALYSIS
CRYSTAL-STRUCTURE
SPACBA PILUS
ADHESION
PURIFICATION
1182 Biochemistry, cell and molecular biology
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