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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.

Original publication

DOI

10.1107/S0907444904026800

Type

Journal article

Journal

Acta Crystallogr D Biol Crystallogr

Publication Date

01/2005

Volume

61

Pages

108 - 111

Keywords

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