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Kumar Megta, Abhin and Palva, Airi and von Ossowski, Ingemar and Krishnan, Vengadesan (2019) SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis. Acta Crystallographica Section F Structural Biology Communications, 75 (12). pp. 731-737. ISSN 2053-230X

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SpaB, an Atypically Adhesive Basal Pilin From the Lactobacillar SpaCBA Pilus Crystallization and X-ray Diffraction Analysis.pdf - Published Version
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The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The crystallization of SpaB remained particularly challenging until the implementation of a three-pronged crystallization approach involving C-terminal tail truncation, surface lysine methylation and magnesium additives. Ultimately, hexagonal crystals of SpaB were produced and were able to diffract to a resolution of 2.4 Å. This crystal form belonged to space group P6522 or P6122, with unit-cell parameters a = b = 51.53, c = 408.22 Å, α = β = 90.0, γ = 120.0°. Obtaining an interpretable electron-density map via single-wavelength anomalous diffraction (SAD) using iodide-derivative data sets did not succeed owing to the weak anomalous signal. As an alternative, attempts to provide phases by molecular replacement using the iodide-SAD data from SpaB and a collection of distant homology models (<28% sequence identity) are in progress.

Item Type: Article
Subjects: Biochemical and Biophysical Sciences
Depositing User: RCB Library
Date Deposited: 14 Mar 2023 09:50
Last Modified: 14 Mar 2023 09:50
URI: http://rcb.sciencecentral.in/id/eprint/253

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