Data-collection strategy for challenging native SAD phasing

Acta Crystallogr D Struct Biol. 2016 Mar;72(Pt 3):421-9. doi: 10.1107/S2059798315024110. Epub 2016 Mar 1.


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.

Keywords: anomalous signal; complex; data-collection strategy; membrane protein; native SAD phasing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Associated Proteins / chemistry
  • Cattle
  • Crystallography, X-Ray / methods*
  • DNA / chemistry
  • Diacylglycerol Kinase / chemistry
  • Escherichia coli / chemistry
  • Escherichia coli / enzymology
  • Models, Molecular
  • Protein Conformation
  • Proteins / chemistry*
  • RNA, Guide, Kinetoplastida / chemistry
  • Rats
  • Staphylococcus / chemistry
  • Staphylococcus / enzymology
  • Stathmin / chemistry
  • Tubulin / chemistry


  • CRISPR-Associated Proteins
  • Proteins
  • RNA, Guide
  • Stathmin
  • Tubulin
  • DNA
  • Diacylglycerol Kinase