Computational protocol: Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

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Protocol publication

[…] Molecular modeling was carried out to predict the structure of peptide KH16p and its interaction with the 14-3-3ζ protein. Peptide structure was prepared using the build option in Maestro and addition of phosphate group was done at S7 (S621 of C-Raf) position of KH16p. The peptide was solvated in orthorhombic water box containing TIP3P water models []. The prepared peptide structure was processed for conformational sampling simulated annealing using OPLS_2005 force-field []. The time frame used for simulated annealing was 5 ns, after which the production run for 15 ns at a constant temperature of 300 K was used for the conformational sampling of explicit solvent medium. Full details of the process are found in a previously published report []. Final structures of peptides were analysed with respect to energy component to select conformation for further studies. Lowest energy structure was adopted for docking process.The peptide and 14-3-3ζ protein model (PDB accession code 4IHL; chain A) [] were subjected to protein preparation wizard [,]. This process helps to avoid potential problems associated with overlapping atoms, bond order, and/or missing atoms. Water molecules present in the X-ray crystal structure were removed during the preparation. Patchdock [] server was used for obtaining the complex of KH16p peptide and 14-3-3ζ protein, with all the default value settings. Validation of the docked complexes was done in conjunction to the STD NMR results []. The obtained complex was then solvated in TIP3P water models in orthorhombic boundary conditions. The adopted protocol and parameters used for minimization and equilibration steps are similar to previous reports []. A production run of 15 ns was set with NTP ensemble using a cut-off value of 10 Å. Trajectory frames were saved at an interval of 5 ps for subsequent analysis. […]

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