Crystal structure of Eco-GluRS
The protein purification and crystallization of an engineeredEco -GluRS (K236E/E328A) was previously reported (27). Since the corresponding wild type GluRS failed to crystallize despite multiple attempts under diverse conditions, GluRS structure was solved using the previously collected data (resolution up to 3.3 Å). The data set was processed using iMosflm (CCP4i, Oxford, UK) and corrected for anisotropy with the STARANISO server (staraniso.globalphasing.org) to perform an anisotropic cutoff. Data collection and processing statistics are summarized in Table 1. The initial phases were determined by the molecular-replacement method using Phaser (28). Molecular-replacement was performed using PDB entry 2cfo (GluRS from T. elongatus ; (6)) that shows 42.8% sequence identity with the Eco -GluRS as search models showed two monomers in the asymmetric unit. Refinement of the atomic coordinates was performed using the CCP4 suite and phenix. During refinement, restraints of torsion-liberation-screw (TLS) groups and Torsion-angle non-crystallographic symmetry (NCS) were applied. The model was further constructed followed by iterative rounds of manual rebuilding in Coot (29) and refinement in REFMAC5 or Phenix.Refine. Structure validation was performed with PROCHECK (30).