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Acta Crystallogr F Struct Biol Commun. 2015 Jan 1;71(Pt 1):103-6. doi: 10.1107/S2053230X14027216. Epub 2015 Jan 1.

Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG.

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Regional Centre for Biotechnology, Gurgaon, Haryana 122 016, India.
Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.


SpaD is the predicted backbone-pilin subunit of the SpaFED pilus, whose loci are encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG, a Gram-positive gut-adapted commensal strain with perceived probiotic benefits. In this study, soluble recombinant SpaD protein was overproduced in Escherichia coli and then purified by Ni2+-chelating affinity and gel-filtration chromatography. After limited proteolysis with α-chymotrypsin, good-quality crystals of SpaD were obtained which diffracted beyond 2.0 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=50.11, b=83.27, c=149.65 Å. For phasing, sodium iodide-derivatized crystals were prepared using the halide quick-soaking method and diffraction data were collected in-house to a resolution of 2.2 Å. An interpretable electron-density map was successfully obtained using single-wavelength anomalous diffraction (SAD).


Lactobacillus rhamnosus GG; SpaFED pili; adhesion; fimbria; in-house iodide SAD phasing; limited proteolysis

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