Crystallization and structural determination of ItaT

For crystallization of the ItaT(G115D) protein, 0.2 μl of the protein solution (2 mg/ml) was mixed with 0.2 μl of reservoir solution, containing 100 mM Tris–HCl, pH 7.6, 30 mM sodium citrate, and 26% (v/v) PEG3350, and the crystals were generated by the hanging drop vapor diffusion method at 20°C. Data sets were collected on beamline 17A at the Photon Factory at KEK, Japan. The crystals were flash cooled in 1.1× concentrated reservoir solution, supplemented with 30% (v/v) ethylene glycol as a cryoprotectant. The data were indexed, integrated, and scaled with XDS (24). The homology model structure of ItaT was built by the SWISS-MODEL server (25) using the K. pneumoniae KacT structure (18), and the initial phase was determined by the molecular replacement method, using the homology model as the search model, by Phaser. The structure was refined with phenix.refine (26), and manually modified with Coot (27).