Molecular dynamics simulations of the E. crus-galli CT domain in complex with metamifop

The CT-metamifop complex was subjected to MD simulations using the SANDER module of the AMBER 14 package³⁶ to obtain a stable conformation. The antechamber module was used to prepare metamifop by the AM1-BCC method^37,38. The pdb4amber program was applied to remove hydrogen atoms from the CT domain, and the program Reduce was used to add hydrogens³⁹. The AMBER ff14SB force field⁴⁰ and GAFF force field⁴¹ were adopted for the CT domain and metamifop, respectively. The complex was solvated in an octahedral box of TIP3P water molecules⁴², which extended 10 Å from any given atoms in the system, and then neutralized by adding Na⁺ ions.

The system was minimized in 2000 steps steepest descent (SD) and 2000 steps conjugate gradient (CG) with the system free and heated gradually from 0 to 300 K using Langevin thermostats with a collision frequency of 1.0 in 50 ps with the force constant of a restraint on all CA, C, N atoms of the complex at 4 kcal·mol⁻¹·Å². Then, the model was relaxed in 4 discrete simulation steps of 250 ps at 300 K and constant pressure (1 atm) by decreasing the force constant of the imposed position restraint on all CA, C, N atoms of the complex from 3 to 0 kcal·mol⁻¹·Å². The MD simulations ran for an additional 3 ns after relaxation at a constant temperature of 300 K with a time constant for heat bath coupling of 5 ps using the weak-coupling method. The Particle Mesh Ewald method⁴³ was selected for periodic long-range electrostatic force and a 10 Å cutoff for nonbonding Van der Waals interactions. All bonds were constrained using the SHAKE algorithm⁴⁴. The leap-frog algorithm⁴⁵ was used with a timestep of 2 fs. Coordinates were recorded every 2 ps during the MD simulations.