The leapfrog integrator with a timestep of 2 fs was used, together with the LINCS 91 al-Figure : Visual representation of the steps in the enhanced sampling methods used in this work. (A) In umbrella sampling, the pathway is obtained by pulling a bound molecule away from its partner. From the pulling trajectory, N frames are selected as reference points for N independent restrained umbrella runs. The final PMF is computed by combining data from all umbrella simulations using the WHAM method. (B) AWH with a single walker. In an AHW simulation, there is no initial path, but sampling of transitions between bound (left) and unbound states (right) is enhanced by dynamically updating a biasing potential at various points along the reaction coordinate, until the trajectory samples a uniform distribution along that path. The points at which such updates occur depend on the sampling history. Therefore, after an update happens at point x 0 , the next updates can occur at both smaller and larger values of the reaction coordinate, avoiding the unidirectionality of US. (C) AWH with multiple walkers. With multiple walkers, several AWH simulations are run in parallel each under the influence of the same single biasing potential, which is updated by all walkers simultaneously. gorithm to constrain lengths of bonds to hydrogen atoms of the solute molecules, and the SETTLE algorithm to constrain internal degrees of freedom of the water molecules. The aforementioned parameters were the same for pulling, umbrella sampling, and AWH simulations. For the additional simulations with the Amber ff99SB-ILDN and for system 5 with the GROMOS96 53A6 force fields, the parameters can be found in the input files provided in the SI.