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Anomalous Dispersion Phasing (anomalous + dispersion_phasing)
Selected AbstractsStructure of Streptococcus agalactiae serine/threonine phosphataseFEBS JOURNAL, Issue 12 2007The subdomain conformation is coupled to the binding of a third metal ion We solved the crystal structure of Streptococcus agalactiae serine/threonine phosphatase (SaSTP) using a combination of single-wavelength anomalous dispersion phasing and molecular replacement. The overall structure resembles that of previously characterized members of the PPM/PP2C STP family. The asymmetric unit contains four monomers and we observed two novel conformations for the flap domain among them. In one of these conformations, the enzyme binds three metal ions, whereas in the other it binds only two. The three-metal ion structure also has the active site arginine in a novel conformation. The switch between the two- and three-metal ion structures appears to be binding of another monomer to the active site of STP, which promotes binding of the third metal ion. This interaction may mimic the binding of a product complex, especially since the motif binding to the active site contains a serine residue aligning remarkably well with the phosphate found in the human STP structure. [source] Use of novel selenomethionine-resistant yeast to produce selenomethionyl protein suitable for structural analysisFEMS YEAST RESEARCH, Issue 3 2009Toshihiko Kitajima Abstract Yeast is widely used to determine the tertiary structure of eukaryotic proteins, because of its ability to undergo post-translational modifications such as glycosylation. A mutant lacking S -adenosylmethionine synthesis has been reported as a suitable host for producing selenomethionine derivatives, which can help solve phase problems in protein crystallography. However, the mutant required external addition of S -adenosylmethionine for cell proliferation. Here, a selenomethionine-resistant Pichia pastoris mutant that showed S -adenosylmethionine autotrophy was isolated. Human lysozyme expressed by the mutant under the control of constitutive promoter contained selenomethionine at 65% occupancy, sufficient for use as a selenomethionine derivative for single-wavelength anomalous dispersion phasing. [source] The magic triangle goes MAD: experimental phasing with a bromine derivativeACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Tobias Beck Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br,K edge was successfully carried out. Radiation damage to the bromine,carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out. [source] Crystallization and X-ray diffraction analysis of the RNA primer/promoter-binding domain of influenza A virus RNA-dependent RNA polymerase PB2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Takashi Kuzuhara The C-terminal domain protein (amino-acid residues 535,759) of the PB2 subunit of the RNA-dependent RNA polymerase from the highly pathogenic influenza A virus was expressed as a soluble protein in Escherichia coli and crystallized using sodium formate as a precipitant. Data sets were collected from crystals of native and selenomethionine-substituted protein on the KEK NW12 beamline at the Photon Factory and the crystals diffracted to a maximum resolution of 2.44,Å for the SeMet-derivative crystal. The native crystals were found to belong to space group P3221, with unit-cell parameters a = b = 52.5, c = 156.3,Å. The Matthews value (VM) was 2.7,Å3,Da,1, assuming the presence of one molecule in the asymmetric unit. The SeMet-derivative crystals were found to belong to the same space group, with unit-cell parameters a = b = 52.6, c = 156.4,Å. Attempts are being made to solve the structure by multi-wavelength anomalous dispersion phasing. [source] | ||||||||||