2.7  Simulations of MEPVC-Innate Immune Receptors
In order to gain insights into the dynamics of the vaccine construct with the receptors, molecular dynamics (MD) simulations have been carried out. The simulation analysis was also important to validate the exposure of epitopes towards the host structure for identification and handling of a substantial outcome. The MD simulations took place in three different phases: system preparation, pre-processing and production [47] with an Assistant model building with Energy Refinement (AMBER) 16 [48]. The antechamber program [49] was used to build the libraries and parameters for the TLR4 and vaccine construct. The TIP3P solvation box (size 12 Å) was inserted to solvate the construct. To study the intermolecular interactions, force field, ff14SB [50] was used whereas, the system was neutralized by the addition of Na+ counter ions. In the second phase of MD simulations, the energy minimization of the complexes was carried out. Each complex was minimized using the following steps: the energy minimization of hydrogen atoms (500 cycles), energy minimization of water box (1000 cycles, control of 200 kcal/mol – Å2 on rest of the system), minimization of the whole atoms of the system (1000 cycles with the restraint of 5 kcal/mol –Å2 on Cα atoms), and the rest of the system was subjected to non-heavy atoms minimization with 300 cycles and restraint of 100 kcal/mol –Å2. In the next step, the system was gradually heated from 0 K to 300 K with the time step of 2 femtoseconds and restraints of 5 kcal/mol –Å2 on Cα atoms. In order to sustain the temperature of the system, Langevin dynamics [51] with the gamma value of 1.0 was castoff. The SHAKE algorithm [52] was used to put constraints on the hydrogen bonds of the system for heating. In the next step, systems were equilibrated for 100 ps with a time step of 2 fs followed by pressure equilibrium, which was attained using the NPT ensemble with restraints of 5 kcal/mol – Å 2 on Cα atoms. The same step was extended for 50 ps with the 1 scale down on restraints on carbon atoms. However, the step of system equilibration was carried out for a time scale of 1 nanosecond followed by the production run of 100 ns with a time scale of 2 fs. For the production run, the Berendsen algorithm [53] with the NVT ensemble cast off with a cut-off of 8.0 Ǻ. Simulation trajectories were calculated to investigate the strength of a complex via the CPPTRAJ module [54] of AMBER. However, the visualization of simulation trajectories was done with UCSF Chimera [55], DS Visualizer [45] and VMD [46].