Computational protocol: Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy

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Protocol publication

[…] The structures of the CTR:CT complexes were generated from the cryo-electron microscopy structure of the calcitonin receptor and the X-ray structure of the CTR ECD . Missing loops were generated using Modeller (1000 structures) and the final loop selected to ensure that the conserved residues faced inwards by analysis of the correlation of the conservation, as measured using the Shannon entropy, and the extent to which a residue is buried . [...] CTR:sCT and CTR:hCT complexes were prepared for molecular dynamics simulations by means of a multistep procedure that integrates both python htmd and tcl (Tool Command Language) scripts. The pdb2pqr and propka software were used to check the protein’s structural integrity and to add hydrogen atoms (configurations of titratable amino acid side chains were visually inspected) appropriate for a simulated pH of 7.0. The CTR was embedded in rectangular matrixes of a 1-palmitoyl-2-oleyl-sn-glycerol-3-phospho-choline (POPC) bilayer (previously built by using the VMD Membrane Builder plugin 1.1; http://www.ks.uiuc.edu/Research/vmd/plugins/membrane) through an insertion method : receptors were first oriented according to the CTR coordinates from the OPM database , then lipids overlapping the protein were removed and TIP3P water molecules were added to the simulation box by means of the VMD Solvate plugin 1.5 (http://www.ks.uiuc.edu/Research/vmd/plugins/solvate). Charge neutrality was finally reached by adding Na+/Cl− counter ions to a final concentration of 0.154 M, according to the VMD Autoionize plugin 1.3 (http://www.ks.uiuc.edu/Research/vmd/plugins/autoionize). The CHARMM36 force field was used. […]

Pipeline specifications

Software tools MODELLER, HTMD, PDB2PQR, PROPKA, VMD
Applications cryo-EM, Protein structure analysis
Organisms Homo sapiens