Computational protocol: Structure function and engineering of multifunctional non-heme iron dependent oxygenases in fungal meroterpenoid biosynthesis

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Protocol publication

[…] The following procedures were all performed under anaerobic conditions. Well-diffracting AusE crystals were obtained at 20 °C, in 20% (w/v) PEG4000, 100 mM sodium citrate (pH 5.6), 10 mM MnCl2, and 15 mg ml−1 of purified AusE with a vapor diffusion method. PrhA, PrhA-V150L/A232S, and PrhA-V150L/A232S/M241V crystals were obtained at 20 °C, in 27% (w/v) PEG3350, 200 mM lithium citrate, and 15 mg ml−1 of purified enzymes with a vapor diffusion method. PrhA crystals were soaked in 25% (w/v) PEG3350 and 200 mM lithium citrate solution (solution A), containing 10 mM FeSO4, 50 mM αKG, and 5 mM 5 for 6 h at 20 °C. PrhA-V150L/A232S crystals were soaked in solution A with 10 mM FeSO4, 50 mM αKG, and 10 mM 5 for 10 h at 20 °C. PrhA-V150L/A232S crystals were soaked in solution A with 10 mM FeSO4, 50 mM αKG, and 5 mM 3, 6, or 7 for 6 h at 20 °C. PrhA-V150L/A232S/M241V crystals were soaked in solution A with 10 mM FeSO4, 50 mM αKG, and 5 mM 3 for 6 h at 20 °C. The crystals were moved into the reservoir solution or the soaking solution with 25% (v/v) glycerol for 10 s for cryoprotection and flash-cooled in liquid nitrogen. BL-1A and BL-17A (Photon Factory, Tsukuba, Japan) were used for obtaining X-ray diffraction data sets. We used wavelengths of 1.8926 Å (Mn absorption edge) for single-wavelength anomalous diffraction (SAD) of wild-type AusE. The other data were collected under the wavelengths of 0.9800 or 1.1000 Å.XDS and AIMLESS were used for data processing and scaling. The Mn substructure determination and the phase calculation were performed by the SAD method with AutoSol in PHENIX, . Initial model of AusE was constructed by AutoBuild in PHENIX, . On the other hand, the initial phases of the PrhA, PrhA-V150L/A232S, and PrhA-V150L/A232S/M241V structures (Phaser in PHENIX) determined by molecular replacement using AusE structure—a search model. Model building was performed by Coot and refined with Phenix.Refine.The three-dimensional models of 3, 5, 6, and 7 were calculated with the Chem3D Ultra 13 (CambridgeSoft). The occupancy of the substrate was maintained at 1.0 during refinements, because the B-factor values of ligand atoms were similar to the surrounding amino acid residues. The final crystal data and intensity statistics are listed in Supplementary Tables , , and . The cavity volumes were predicted using CASTP server (http://cast.engr.uic.edu/cast/). PyMOL (http://www.pymol.org) was used to prepare all structure representations. […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, CASTp, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Fungi, Aspergillus nidulans
Chemicals Spironolactone