Open AccessCrystallization of leucyl‐tRNA synthetase complexed with tRNA Leu from the archaeon Pyrococcus horikoshii
Author(s) -
Fukunaga Ryuya,
Ishitani Ryuichiro,
Nureki Osamu,
Yokoyama Shigeyuki
Publication year - 2005
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309104021827
Subject(s) - pyrococcus horikoshii , transfer rna , aminoacyl trna synthetase , crystallization , chemistry , biochemistry , genetics , rna , biology , enzyme , gene , organic chemistry
All five tRNA Leu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl‐tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNA Leu isoacceptors were attempted. Cocrystals were obtained by the hanging‐drop vapour‐diffusion method, but only when the tRNA Leu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNA Leu , suggesting that they are LeuRS–tRNA Leu complex crystals. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P 2 1 2 1 2, with unit‐cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is expected to contain two complexes of LeuRS–tRNA Leu , with a corresponding crystal volume per protein weight of 2.9 Å 3 Da −1 and a solvent content of 57.3%.