The Robetta server [45 (link),46 (link)] was utilized for molecular modeling of the E protein as a dimer since this is the conformation that acts in membrane fusion, and it was expected to be more stable than the monomer. The genome sequence submitted to the server was retrieved from GenBank (accession number MH932545), and after modeling, the model protein was submitted to MolProbity [47 (link)] for quality analysis.
After validation of the model, the histidine pronation state for pH 7.4 was predicted using the PropKa server [48 (link)], and the initial steps for molecular dynamics (MD) were performed using GROMACS 5.1.2 [49 (link)] and the AMBER99SB-ILDN force field [50 (link)]. The MD protocol described here was previously executed in other studies [51 (link),52 (link),53 (link)]. The initial structure was inserted into a cubic box with a minimum distance of 10 angstroms (Å) between each box edge and the protein atom. This box was solvated with TIP3P water, and the system was neutralized with sodium ions.
The SETTLE algorithm [54 (link)] was used to maintain the internal rigid structure of the solvent molecules (water), while the solute covalent bonds involving hydrogen atoms were constrained by the LINCS algorithm [55 (link)]. The system temperature was set to 310 K (36.85 °C) and the pressure to 1 atm; both parameters were controlled by the V-rescale and Parrinello–Rahman algorithms [56 (link),57 (link)], respectively. A cutoff of 1.0 nm was defined for nonbonded interactions, and the Ewald summation method for particle networks was used for long-range electrostatic interactions. The leapfrog algorithm was used to integrate the MD equations of motion with two fs as the time step.
Initially, the system was subjected to two energy minimization steps. The first was performed with 500 steps of the steepest descent algorithm and protein position restriction. The second minimization step utilized the same algorithm but with 10,000 steps and flexible water. After the minimization steps, the system was subjected to equilibration steps consisting of two 100 picosecond (ps) phases: an NVT ensemble and an NPT ensemble with protein position restriction for equilibration of the thermodynamic variables. The third and final equilibration phase was performed as an NPT ensemble of 1 nanosecond (ns) without protein restriction. Finally, the production run was performed as an NPT ensemble at 310 K with a total time of 100 ns. Trajectory analysis was performed using GROMACS 5.1.2. This work was performed in replicates (three replicates) to obtain a range of different conformations from independent MD simulations.
After validation of the model, the histidine pronation state for pH 7.4 was predicted using the PropKa server [48 (link)], and the initial steps for molecular dynamics (MD) were performed using GROMACS 5.1.2 [49 (link)] and the AMBER99SB-ILDN force field [50 (link)]. The MD protocol described here was previously executed in other studies [51 (link),52 (link),53 (link)]. The initial structure was inserted into a cubic box with a minimum distance of 10 angstroms (Å) between each box edge and the protein atom. This box was solvated with TIP3P water, and the system was neutralized with sodium ions.
The SETTLE algorithm [54 (link)] was used to maintain the internal rigid structure of the solvent molecules (water), while the solute covalent bonds involving hydrogen atoms were constrained by the LINCS algorithm [55 (link)]. The system temperature was set to 310 K (36.85 °C) and the pressure to 1 atm; both parameters were controlled by the V-rescale and Parrinello–Rahman algorithms [56 (link),57 (link)], respectively. A cutoff of 1.0 nm was defined for nonbonded interactions, and the Ewald summation method for particle networks was used for long-range electrostatic interactions. The leapfrog algorithm was used to integrate the MD equations of motion with two fs as the time step.
Initially, the system was subjected to two energy minimization steps. The first was performed with 500 steps of the steepest descent algorithm and protein position restriction. The second minimization step utilized the same algorithm but with 10,000 steps and flexible water. After the minimization steps, the system was subjected to equilibration steps consisting of two 100 picosecond (ps) phases: an NVT ensemble and an NPT ensemble with protein position restriction for equilibration of the thermodynamic variables. The third and final equilibration phase was performed as an NPT ensemble of 1 nanosecond (ns) without protein restriction. Finally, the production run was performed as an NPT ensemble at 310 K with a total time of 100 ns. Trajectory analysis was performed using GROMACS 5.1.2. This work was performed in replicates (three replicates) to obtain a range of different conformations from independent MD simulations.
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