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Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain

Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using... The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatized crystals suitable for structural determination. Unlike in previous reports, a standard autotrophic expression strain was used and only single-wavelength anomalous dispersion (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. The native protein crystallized in two different crystal forms (form 1 belonging to space group C2221 and form 2 belonging to space group C2), which diffracted to 2.4 and 2.0 A resolution, respectively, whilst Se-derivatized protein crystallized in space group C2 and diffracted to 1.7 A resolution. The Se-derivatized crystals are suitable for SAD structure determination using only the anomalous signal derived from the SeCys residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Crystallographica Section D: Biological Crystallography International Union of Crystallography

Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain

Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain


Abstract

The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatized crystals suitable for structural determination. Unlike in previous reports, a standard autotrophic expression strain was used and only single-wavelength anomalous dispersion (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. The native protein crystallized in two different crystal forms (form 1 belonging to space group C2221 and form 2 belonging to space group C2), which diffracted to 2.4 and 2.0 A resolution, respectively, whilst Se-derivatized protein crystallized in space group C2 and diffracted to 1.7 A resolution. The Se-derivatized crystals are suitable for SAD structure determination using only the anomalous signal derived from the SeCys residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.

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References (19)

Publisher
International Union of Crystallography
Copyright
Copyright (c) 2011 International Union of Crystallography
Subject
selenocysteine, SAD, non-auxotrophic Escherichia coli strains, CsgC
ISSN
0907-4449
eISSN
1399-0047
DOI
10.1107/S0907444910042022
pmid
21206057
Publisher site
See Article on Publisher Site

Abstract

The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatized crystals suitable for structural determination. Unlike in previous reports, a standard autotrophic expression strain was used and only single-wavelength anomalous dispersion (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. The native protein crystallized in two different crystal forms (form 1 belonging to space group C2221 and form 2 belonging to space group C2), which diffracted to 2.4 and 2.0 A resolution, respectively, whilst Se-derivatized protein crystallized in space group C2 and diffracted to 1.7 A resolution. The Se-derivatized crystals are suitable for SAD structure determination using only the anomalous signal derived from the SeCys residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.

Journal

Acta Crystallographica Section D: Biological CrystallographyInternational Union of Crystallography

Published: Dec 16, 2010

Keywords: selenocysteine ; SAD ; non-auxotrophic Escherichia coli strains ; CsgC .

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