4.4. Running SepTop in GROMACS. All MD simulations were performed using GRO-MACS 2021.2. For simulations in the binding site, we used an alchemical path with a total of 20 λ windows, 8 states for leg B, 5 for leg C and 8 for leg D. Leg B and leg C were run in one step, meaning that a single topology file and therefore only two end states were used for leg B and leg C together. This combined step then used 12 λ windows, making this a total of 20 λ windows. The details of the alchemical path can be found in the free energy calculations section of the MDP files provided in the SI.