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).