Computational protocol: Residues Controlling Facial Selectivity in an AlkeneReductase and Semirational Alterations to Create StereocomplementaryVariants

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Protocol publication

[…] Diffraction data were collected using synchotron radiation under cryogenic conditions. Reflection data for the Tyr78Trp OYE 2.6 were processed by an in-house program to yield a merged set suitable for refinement; those for the Tyr78Trp/Ile113Cys OYE 2.6 were processed using the HKL3000 program suite. Phases were obtained by molecular replacement using the AutoMR utility of PHENIX using the wild-type P. stipitis OYE 2.6 (PDB code 3TJL) as the search model (after removing FMN, water molecules, and nonidentical side chains). The initially calculated 2F0–Fc and F0–Fc maps showed easily identifiable electron density patterns for the FMN, which served as a validation for the molecular replacement solution. Iterative cycles of refinement and model building, with PHENIX and COOT, respectively, were performed until the error statistics reached acceptable levels. At this stage of refinement, the active site contained areas of unaccounted-for electron density. Potential ligand structures were built using proDrg and then incorporated into the model for further refinement. All protein structure figures were created using PyMOL (Schrödinger, LLC). […]

Pipeline specifications

Software tools PHENIX, Coot, PRODRG, PyMOL
Applications Drug design, Protein structure analysis
Organisms Scheffersomyces stipitis