Computational protocol: Determination of the Crystal Structure and Active Residues of FabV, the Enoyl-ACP Reductase from Xanthomonas oryzae

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Protocol publication

[…] SeMet-xoFabV protein crystals were grown using the hanging-drop vapour diffusion method in a reservoir solution of 100 mM Bis-Tris (pH 6.5) containing 200 mM NaCl, 1 mM DTT and 15% PEG 3350. Crystals appeared in 24 h and were of high quality. Two diffraction datasets were collected. The HKL-2000 package was used for data processing. The crystal belonged to space group P212121, with cell dimensions of a = 50.52 Å, b = 74.53 Å and c = 107.39 Å. PHENIX was used for two-wavelength MAD phasing and automatic model building. One xoFabV monomer was found in the asymmetric unit, and five out of the six expected selenium atoms were located in each monomer. Several iterative rounds of manual building in COOT and refinement in PHENIX were performed until acceptable Rwork (18.5%) and Rfree (20.5%) values were achieved. The stereochemical quality of the final model at 1.6 Å was validated using PROCHECK , which showed that 93.2% of the residues are within the most favoured region of the Ramachandran plot and 6.8% are within the additional allowed region. The Matthews coefficient was 2.1 Å3 Da−1, and the solvent content was 44.2%. […]

Pipeline specifications

Software tools HKL-2000, PHENIX, Coot, PROCHECK
Application Protein structure analysis
Organisms Xanthomonas oryzae, Escherichia coli, Burkholderia mallei, Vibrio cholerae
Chemicals Tyrosine