Going soft and SAD with manganese.
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.
Salgado
Paula S
PS
Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN, England.
Walsh
Martin A
MA
Laurila
Minni R L
MR
Stuart
David I
DI
Grimes
Jonathan M
JM
D020816:Amino Acid Motifs; D001331:Automation; D017126:Bacteriophage phi 6 / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002412:Cations; D003461:Crystallography; D018360:Crystallography, X-Ray / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D012984:Software; D014961:X-Ray Diffraction; D014965:X-Rays
D002412:Cations; D011506:Proteins; D008345:Manganese; D012321:DNA-Directed RNA Polymerases; D002118:Calcium