Crystallization and X-ray diffraction analysis of the RNA primer/promoter-binding domain of influenza A virus RNA-dependent RNA polymerase PB2.
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 A for the SeMet-derivative crystal. The native crystals were found to belong to space group P3(2)21, with unit-cell parameters a = b = 52.5, c = 156.3 A. The Matthews value (V(M)) was 2.7 A(3) 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 A. Attempts are being made to solve the structure by multi-wavelength anomalous dispersion phasing.
Kuzuhara
Takashi
T
Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan. kuzuhara@ph.bunri-u.ac.jp
Kise
Daisuke
D
Yoshida
Hiroko
H
Horita
Takahiro
T
Murazaki
Yoshimi
Y
Utsunomiya
Hiroko
H
Tsuge
Hideaki
H
D001665:Binding Sites / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
C057027:PB2 protein, Influenzavirus A; D010446:Peptide Fragments; C034477:RNA primers; D012367:RNA, Viral; D014764:Viral Proteins; D012313:RNA; D012324:RNA-Dependent RNA Polymerase