Computational protocol: Structural basis for the activation of acid ceramidase

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Protocol publication

[…] The structure of nmr aCDase was solved by iodine single-wavelength anomalous diffraction using Autosol in Phenix, manually built in Coot and refined against a dataset from a non-soaked crystal. The structures of human and cmw enzymes were obtained by molecular replacement using Phaser in Phenix. Refinement was carried out by phenix.refine with the following parameters: anisotropic non-hydrogen B-factors for nmr aCDase, non-crystallographic symmetry restraints for its two homologs, translation-libration-screw for the human-aCDase. Crystallographic data collection and refinement statistics are presented in Table . Structural images were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC). No density for ceranib-2 was observed in the electron density of human-aCDase. Calculation of protein side chain pKa values was carried out using the PDB2PQR server. [...] The coordinates for various initial orientations of a C12 ceramide molecule were manually docked into the active site of the human-aCDase structure in Coot. The distance between the Cys 143 sulfur atom and the ceramide carbonyl carbon was fixed at 3 Å. Residues near the active site were fixed to improve the stability of the energy minimization procedure using Groningen Machine for Chemicals Simulations (GROMACS) 4.6.2 package with GROMOS 96 force field. The aCDase topology file was generated by using the GROMOS 53A6 force field. The ceramide topology file was generated with the same force-field as the protein by the Automated Topology Builder Server. Each aCDase-ceramide complex was placed and solvated in the center of a dodecahedron box with an adjusted 10 Å distance from each side of the box. The system’s charge was neutralized by the addition of a single chloride ion. The energy minimization for each system (nsteps = 50 000) was done by the steepest descent algorithm. The Particle Mesh Ewald method was used to calculate electrostatic interactions. The cut-off distances for the Van der Waals interactions, the Coulomb interactions, and the short-range neighbor list were all set to 10 Å. […]

Pipeline specifications

Software tools PHENIX, Coot, PyMOL, PDB2PQR, GROMACS
Application Protein structure analysis
Organisms Homo sapiens
Diseases Epilepsies, Myoclonic, Neoplasms, Farber Lipogranulomatosis
Chemicals Sphingosine