Computational protocol: Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid

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Protocol publication

[…] After the release of the primary sequences of DNA and RNA polymerases from M. perniciosa mitochondrial plasmid, they are available in the Witches' broom project database (LGE). 3D models were built by Comparative Modeling approach. Initially, both DNA and RNA polymerase sequences were subjected to the BLASTp algorithm [] restricted to the Protein Data Bank (PDB). The templates found were aligned with the protein sequences of both DNA and RNA polymerases by TCOFFEE [] to find conserved regions and motifs. The 3D models were constructed using SwissPdb Viewer 3.7 [] following a standard protocol: (I) load template pdb file; (II) align primary target sequence with template; (III) submit modeling request to Swiss Model Server. Then, the initial models constructed by SwissPdb Viewer were prepared using LEAP and submitted to SANDER for structure refinement. The model structures were fully minimized with 100 steps of steepest descent followed by 100 more steps of conjugate gradient to an RMS gradient of 0.01 kcal/2.71Å in vacuum, and then in water for 200 steps of steepest descent followed by 200 more steps of conjugate gradient to an RMS gradient of 0.01 kcal/2.71Å. Next, MD simulations of the refined structures were performed in water using f99 force field at 300 K for 2000 ps. All MD simulations were carried out without constrain methods. The cutoff value of 14 Å was used for minimization of geometry and MD simulations. LEAP and SANDER are utilities of AMBER 9.0 [,]. Additionally, all calculations were performed without restraints. Time averaged structures were generated by time averaging of simulations from the point of a stable trajectory, which was obtained through the end of simulation. The Visual Molecular Dynamics (VMD) software [] was used to visualize trajectory results produced by the SANDER module. Finally, PROCHECK 3.4 [] and Atomic Non-Local Environment Assessment (ANOLEA) [,] were used to evaluate both DNA and RNA polymerases using a Ramachandran plot [] and energy calculations on a protein chain of each heavy atom in the molecule, respectively []. Graphics of RMS × Time were generated by VMD 1.8.6 [] […]

Pipeline specifications

Software tools BLASTP, T-Coffee, VMD, PROCHECK, ANOLEA
Applications Protein structure analysis, Protein physicochemical analysis
Organisms Moniliophthora perniciosa, Theobroma cacao
Diseases Mycoses, Genetic Diseases, Inborn