Going soft and SAD with manganese

Acta Crystallogr D Biol Crystallogr. 2005 Jan;61(Pt 1):108-11. doi: 10.1107/S0907444904026800. Epub 2004 Dec 17.

Abstract

SAD phasing has been revisited recently, with experiments being carried out using previously unconventional sources of anomalous signal, particularly lighter atoms and softer X-rays. A case study is reported using the 75 kDa RNA-dependent RNA polymerase of the bacteriophase phi6, which binds a Mn atom and crystallizes with three molecules in the asymmetric unit. X-ray diffraction data were collected at a wavelength of 1.89 A and although the calculated anomalous signal from the three Mn atoms was only 1.2%, SHELXD and SOLVE were able to locate these atoms. SOLVE/RESOLVE used this information to obtain SAD phases and automatically build a model for the core region of the protein, which possessed the characteristic features of the right-hand polymerase motif. These results demonstrate that with modern synchrotron beamlines and software, manganese phasing is a practical tool for solving the structure of large proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Automation
  • Bacteriophage phi 6 / chemistry
  • Calcium / chemistry
  • Cations
  • Crystallography
  • Crystallography, X-Ray / methods*
  • DNA-Directed RNA Polymerases / chemistry
  • Escherichia coli / metabolism
  • Manganese / chemistry*
  • Models, Molecular
  • Molecular Structure
  • Protein Conformation
  • Proteins / chemistry
  • Software
  • X-Ray Diffraction
  • X-Rays

Substances

  • Cations
  • Proteins
  • Manganese
  • DNA-Directed RNA Polymerases
  • Calcium