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Data-collection strategy for challenging native SAD phasing.

  • Olieric V Swiss Light Source, Paul Scherrer Institut, Villigen PSI, Switzerland.
  • Weinert T Swiss Light Source, Paul Scherrer Institut, Villigen PSI, Switzerland.
  • Finke AD Swiss Light Source, Paul Scherrer Institut, Villigen PSI, Switzerland.
  • Anders C Department of Biochemistry, University of Zurich, Zurich, Switzerland.
  • Li D Membrane Structural and Functional Group, Schools of Medicine and Biochemistry and Immunology, Trinity College, Dublin, Ireland.
  • Olieric N Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, Villigen PSI, Switzerland.
  • Borca CN Swiss Light Source, Paul Scherrer Institut, Villigen PSI, Switzerland.
  • Steinmetz MO Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, Villigen PSI, Switzerland.
  • Caffrey M Membrane Structural and Functional Group, Schools of Medicine and Biochemistry and Immunology, Trinity College, Dublin, Ireland.
  • Jinek M Department of Biochemistry, University of Zurich, Zurich, Switzerland.
  • Wang M Swiss Light Source, Paul Scherrer Institut, Villigen PSI, Switzerland.
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  • 2016-03-10
Published in:
  • Acta crystallographica. Section D, Structural biology. - 2016
anomalous signal
complex
data-collection strategy
membrane protein
native SAD phasing
Animals
CRISPR-Associated Proteins
Cattle
Crystallography, X-Ray
DNA
Diacylglycerol Kinase
Escherichia coli
Models, Molecular
Protein Conformation
Proteins
RNA, Guide
Rats
Staphylococcus
Stathmin
Tubulin
English Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.
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  • English
Open access status
hybrid
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https://sonar.ch/global/documents/263412
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