Protein crystallization, structure determination and refinement

Crystallization was performed at 16°C, using the hanging drop vapor diffusion method. For crystallization, LRS was concentrated to 8mg/ml. Protein solution (1 μl) was mixed with an equal volume of the reservoir solution, consisting of 20% (w/v) PEG 6000, 100 mM Tris–Cl, pH 8.0 and 200 mM Lithium chloride. The crystals were frozen in liquid nitrogen after transferring for a few seconds in the mother liquid which contained 15% glycerol (v/v) as a cryoprotectant.

All crystal diffraction data sets were collected at the Shanghai Synchrotron Radiation Facility beamlines (SSRF, Shanghai, China) BL-19U1 and BL-17U1. The diffraction data were processed using the HKL2000 program package (49). Further data analysis was performed with the CCP4 suite (50). The structure of LRS was initially solved by molecular replacement using PHASER (51) with the structure of aminoacylation domain and C-terminal domain of Pyrococcus horikoshii LRS (PDB ID: 1WZ2) and the structure of isolated hcLRS-CP1 as starting models, and was further improved by manual adjustments using COOT (52). Next, the model was refined using REFINE program in the PHENIX suite (53). The quality of the final model was evaluated using MOLPROBITY (http://molprobity.biochem.duke.edu/). Figures were drawn using PyMOL (http://www.pymol.org/). A structure-based multiple amino acid sequence alignment of LRSs from model organisms was generated using ESPript (54). The parameters for data collection and structure refinements are shown in Supplementary Table S1. The PDB IDs are 6LPF and 6LR6.