Screening orthologs as an important variable in crystallization: preliminary X-ray diffraction studies of the tRNA-modifying enzyme S-adenosyl-methionine:tRNA ribosyl transferase/isomerase.
The genes encoding the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyl transferase/isomerase (QueA) from 12 eubacterial sources were overexpressed in Escherichia coli and the resulting products were purified to homogeneity and subjected to crystallization trials. Using the hanging-drop vapour-diffusion method, crystals suitable for X-ray diffraction experiments were only obtained for the queA gene product from Bacillus subtilis. The crystals belong to the space group P422, with unit-cell parameters a = b = 100.7, c = 150.9 A. Using highly focused synchrotron radiation from the EMBL/ESRF beamline ID13 (Grenoble, France), diffraction to at least 3.2 A could be achieved. A selenomethionyl derivative of the protein was prepared and crystallized for future multiwavelength anomalous diffraction (MAD) experiments.
Grimm
C
C
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35032, Marburg, Germany.
Klebe
G
G
Ficner
R
R
Reuter
K
K
D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007535:Isomerases; D010430:Pentosyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D010430:Pentosyltransferases; C082572:S-adenosylmethionine - tRNA ribosyltransferase-isomerase; D007535:Isomerases