Crystallization of leucyl-tRNA synthetase complexed with tRNALeufrom the archaeonPyrococcus horikoshii

Abstract

All five tRNALeu 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 tRNALeu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNALeu, suggesting that they are LeuRS-tRNALeu complex crystals. A data set diffracting to 3.3 A resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 A. The asymmetric unit is expected to contain two complexes of LeuRS-tRNALeu, with a corresponding crystal volume per protein weight of 2.9 A3 Da(-1) and a solvent content of 57.3%.