Computational protocol: Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease-Associated “Toggle” Calcium-Sensing Receptor Mutations

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Protocol publication

[…] CLUSTALW was used to align the sequences of the human CaSR ECD (residues 25–530) and the mGluR1 ECD . The structure of the ECD of CaSR was modeled based on the crystal structure of mGluR1 (1EWT, 1EWK and 1ISR) using SWISS-MODEL and MODELLER software , and the potential Ca2+-binding sites in the CaSR ECD were predicted using MetalFinder , The electrostatic potentials were calculated using Pymol.MD simulation provides an approach complementary to experiments in live cells for understanding biomolecular structure, dynamics, and function. The initial coordinates for all the simulations were taken from a 2.20 Å resolution x-ray crystal structure with PDBID 1EWK . The AMBER 10 suite of programs was used to carry out all of the simulations in an explicit TIP3P water model , using the modified version of the all-atom Cornell et. al. force field and the re-optimized dihedral parameters for the peptide ω-bond . An initial 2 ns simulation was performed using NOE restraint during the equilibration in order to reorient the side chains of residues in the Ca2+-binding site, but no restraints were used during the actual simulation. A total of three MD simulations were carried out for 50 ns each on the apo-form and the ligand-loaded forms. During the simulations, an integration time step of 0.002 ps was used to solve the Newton's equation of motion. The long-range electrostatic interactions were calculated using the Particle Mesh Ewald method, , and a cutoff of 9.0 Å was applied for non-bonded interactions. All bonds involving hydrogen atoms were restrained using the SHAKE algorithm . The simulations were carried out at a temperature of 300 K and a pressure of 1 bar. A Langevin thermostat was used to regulate the temperature with a collision frequency of 1.0 ps-1. The trajectories were saved every 500 steps (1ps). The trajectories were analyzed using the ptraj module in AMBER 10. […]

Pipeline specifications

Software tools Clustal W, PyMOL
Application Small-angle scattering
Diseases Genetic Diseases, Inborn
Chemicals Amino Acids, Calcium