Umbrella sampling.

To calculate the free energy profile of binding of IFM to its binding site, umbrella sampling was employed using the distance between the COM of F1486 and the COM of N1659 as a reaction coordinate. Each umbrella sampling window was 1 Å wide, and the position of the IFM particle was restrained by applying a harmonic potential on the reaction coordinate. A brief 100-ps-long NPT equilibration was conducted with 1 atm pressure maintained by Berendsen pressure coupling and 300 K, controlled by Berendsen thermostat. This was followed by 10 ns of production simulation in each window. The N1659 residue (located in the IFM particle binding site) was restrained throughout the simulation with a restraining potential force constant of 1,000 kJ ⋅ mol⁻¹ ⋅ nm⁻². The weighted histogram analysis method (WHAM) (86) in GROMACS (gmx wham) was used to combine data from all the umbrella sampling windows to compute the free energy profile.