Aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNAArg (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent.
For crystallization of the ArgRS:tRNAArg complex, protein and tRNA were mixed at a 1:1.3 molar ratio and crystallized in hanging drops. Reservoirs contained 0.1 M Na citrate and 1.95 M (NH4)2SO4 at pH 5.6. Di-pyramid shaped crystals measuring 0.2 × 0.2 × 0.6 mm3 were grown over 2–3 days. All crystals were flash-frozen in liquid nitrogen for x-ray diffraction data collection at 100° K. For the ArgRS:tRNAArg complex, data were recorded at an Quantum 315r charge-coupled device detector (ADSC, Poway, CA) at beamline 19-ID of the Advanced Photon Source.
The space group of EcoArgRS:tRNA crystals is P65, which could not be determined until solving the phase problem. For phasing data of ArgRS:tRNA crystals, molecular replacement was performed with PHENIX.phaser. The structure of SceArgRS in complex with tRNA was employed as the search model. The space group P65 provided a reasonable phase solution. The final model was manually adjusted in COOT and refined with PHENIX.
The solved structure demonstrates several determinant interactions between tRNA and the synthetase in the D-stem of the tRNA. The E coli ArgRS is an alpha-helix rich (about 60%) structure having an active site built on a Rossmann beta-sheet scaffold. The tRNA spreads over the active site with the 3’ end spanding over Ins-2 and D-loop over Add-1 and the anticodon arm pointing toward Add-2.