Computational protocol: The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

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Protocol publication

[…] The AmPDH structure was determined by molecular replacement using the fully automated BALBES pipeline as implemented in CCP4i , , starting with diffraction and sequence data. Automated search model generation in BALBES tested five template models: 2JBV, 1CF3, 1GPE, 1JU2, 1KDG; with sequence identities to PDH of 26.1, 27.0, 25.3, 25.3, and 21.5%, respectively. The best Q value after all model calculations (0.5395) was obtained for 2JBV, which resulted in a suggested structure of 82% probability of being correct. After the integrated refinement with REFMAC5 , the initial R/Rfree values of 0.539/0.538 for this model decreased to 0.448/0.491. Following structure solution, the resulting partially refined model from BALBES was submitted for automated model building using the online ARP/wARP web service available at the EMBL Hamburg website (http://www.embl-hamburg.de/ARP). Automatic model building resulted in a nearly complete model of 554 assigned residues (of 577) in eight chains and R/Rfree values of 0.225 and 0.269, respectively. Refinement was performed using REFMAC5 , , including anisotropic scaling, calculated hydrogen scattering from riding hydrogen atoms. Individual anisotropic B-factor refinement was performed for all atoms. Atomic displacement parameter refinement was performed using the translation, libration, screw-rotation (TLS) model. Eight TLS groups were defined, as determined by the TLS Motion Determination server, TLSMD . Rebuilding and model manipulation were done using O and Coot , guided by σA-weighted 2Fo–Fc and Fo–Fc electron-density maps. Model validation was performed using MolProbity . The 5000 K simulated annealing omit map shown in was calculated using PHENIX . Data collection and model refinement statistics are given in . All pictures showing structures were generated with PyMOL™ (DeLano Scientific LLC, www.pymol.org). The atomic coordinates and structure factors (code 4H7U) for the AmPDH model have been deposited with the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers (http://www.rcsb.org). […]

Pipeline specifications

Software tools REFMAC5, ARP/wARP, Coot, MolProbity, PHENIX, PyMOL
Application Protein structure analysis
Diseases Pyruvate Dehydrogenase Complex Deficiency Disease
Chemicals Glucose, Oxygen, Superoxides, Halogenated Diphenyl Ethers