Computational protocol: A new crystal form of Aspergillus oryzae catechol oxidase and evaluation of copper site structures in coupled binuclear copper enzymes

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Protocol publication

[…] Crystals were harvested and plunged into liquid nitrogen. Ethylene glycol (30%) served as a cryo protectant. Two different data sets (met/deoxy and deoxy) from two crystals were collected. The diffraction data of the met/deoxy crystal were collected at Diamond Light Source (DLS), Oxfordshire, England at MX beamline i02. The diffraction data of the deoxy crystal were collected at the European Synchrotron Radiation Facility (ESRF), Grenoble, beamline ID23-2. Both data sets were processed with the XDS software package []. Data collection statistics are shown in .The crystal structures were solved by the molecular replacement method, and a structure of full-length AoCO4 (PDB code 4J3P) served as a template. Phaser [] software from the CCP4 suite, version 6.5 [], was used in molecular replacement. Coot [] software was used for model building, and the structures were refined with phenix.refine, version 1.11.1–2575 [–]. Anomalous difference Fourier maps for met/deoxy and deoxy data were calculated and used for examining the Cu positions (Figure E in ). For deoxy data, the anomalous signal was too weak and not clearly detectable. Explicit riding hydrogen atoms were refined in the met/deoxy structure. presents the statistics of structure refinement. Copper-histidine restraints with an ideal distance of 2.02 Å and variance of 0.1 Å were used in the refinement. No links between copper ions and oxygen moieties were used to avoid bias in the copper site structure. Matthews_coef, a program in the CCP4 package, determined the solvent content of crystals. The solvent content for the met/deoxy structure was approximately 44% (the Matthews coefficient was 2.2 Å3Da-1) and 45% for the deoxy structure (the Matthews coefficient was 2.3 Å3Da-1). The space group was verified to be P1 using Zanuda software from CCP4 package []. The final refined structures were also run through the online Diffraction Precision Index (DPI) server []. Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5OR3 and 5OR4. [...] The structure factors and coordinate files were downloaded in Coot software through PDB_REDO interface, which automatically downloads the optimized models and electron density maps. All available CBC protein structures were inspected, and the copper sites of the following structures were then further re-refined: 1BT1, 1BT3, 2AHL, 2P3X, 2ZMZ and 4J6T. Initially, water molecules or oxygen species between coppers were totally omitted, and new electron density maps were calculated. REFMAC5 [] from the CCP4 [] software package was used in refinement. Based on the calculated omit electron density maps, some of the structures were re-refined. No links between copper ions and oxygen moieties were used in the refinement. […]

Pipeline specifications

Software tools XDS, CCP4, Coot, PHENIX, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Aspergillus oryzae
Diseases Protein Deficiency
Chemicals Copper, Oxygen