Computational protocol: Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue

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Protocol publication

[…] Crystallization of the CYP107W1-oligomycin A bound complex was carried out as previously described with some modifications (). Briefly, the sitting-drop vapor-diffusion method was employed for the initial crystallization at 14°C, using a Hydra II e-Drop automated pipetting system (Matrix) and the Index, Crystal Screen, Crystal Screen Cryo, Crystal Screen Lite, PEGRx, SaltRx, PEG/Ion, Wizard, and Wizard precipitant synergy screening kits. To determine the structure of the oligomycin A complex, the native CYP107W1 enzyme was co-crystallized with oligomycin A in a 1:10 molecular ratio. Crystals of the oligomycin A complex appeared in Wizard precipitant synergy D2 conditions, with 3.35% (v/v) isopropanol and 1.34 M ammonium citrate/citric acid (pH 6.5).The initial crystals were reproduced in the same conditions by the hanging-drop method, in which drops consisted of 1.0 μl protein solution mixed with 1.0 μl reservoir solution. X-ray diffraction data were collected from the cryoprotected crystal (at 100 K) with 1° rotation at a crystal-to-detector distance of 250 mm using an ADSC Q270 detector at beamline 7A-SBI of the Pohang Light Source (PLS, Pohang, Korea). Diffraction data were integrated and scaled using the HKL-2000 program package (). [...] The structure of CYP107W1 complexed with oligomycin A (CYP107W1-OliA) was solved by molecular replacement with Molrep (), using the native CYP107W1 ligand-free structure (PDB ID: 4WPZ). One monomer of CYP107W1-OliA was present in the asymmetric unit. After refinement of the protein model, the resulting map was examined for the bound ligand density. The oligomycin A ligand model from the PRODRG2 server () was fitted into the electron-density map with Coot (). The overall structure and volume of the substrate binding pocket of CYP107W1-OliA were calculated and generated in PyMol (). Data collection statistics are provided in . […]

Pipeline specifications

Software tools HKL-2000, Molrep, PRODRG, Coot, PyMOL
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Streptomyces avermitilis
Chemicals Azoles, Heme