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 ( 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, 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 ( […]

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