Computational protocol: Atomic resolution structure of serine protease proteinase K at ambient temperature

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Protocol publication

[…] Each structure for SFX and SRX was determined by difference Fourier synthesis using a search model (PDB code: 4b5l). Manual model revision was performed using the Coot program. The program PHENIX was used for structure refinement. Water molecules were incorporated where the difference in density exceeded 3.0σ above the mean and the 2mFo–DFc map showed a density of more than 1.0σ. All reflections were included with no σ cutoff; 5% of the data were randomly selected and omitted during refinement for cross validation by means of the free R-factor. The occupancy of the major conformation was refined first, and then the minor conformation was assigned and refined based on its mFo–DFc map. Anisotropic B-factor refinement was performed, and finally hydrogen atoms were automatically added to the models. Hydrogen atoms were included in the protein and glycerol atoms but not in the nitrate/solvent atoms. The hydrogen omit maps were created by deleting all the hydrogen atoms from the model and refined through PHENIX.The quality of the final model was assessed using PROCHECK and RAMPAGE. The CCP4 package was used for the manipulation of data and coordinates. The electron density maps and structural images were generated using PyMOL. The comparisons of water molecules were performed by WATERTIDY implemented in CCP4. The coordinates and observed intensities of proteinase K have been deposited in the PDBj (accession code 5kxu for SFX and 5kxv for SRX). The total absorbed dose was calculated by RADDOSE. Details of data collection and refinement statistics are shown in . […]

Pipeline specifications

Software tools Coot, PHENIX, PROCHECK, CCP4, PyMOL
Application Protein structure analysis
Chemicals Hydrogen