If the binding mode of a ligand is unknown, we have found it to be helpful to run Sep-Top between different poses to determine the more favorable binding pose which we then used for further simulations (Section 5.4). We also found that adapting the alchemical protocol by modifying bonded and non-bonded interactions of the ligand (e.g. scaling down the LJϵ (ϵ-HREX), and scaling down dihedral force constants) may not be sufficient to sample the correct binding mode sufficiently when starting simulations from the wrong pose (Section 5.4). Based on our results so far, we strongly advise against trying to engage in ligand binding pose prediction while doing free energy calculations, but instead selecting one binding pose prior to the calculation (e.g. by running SepTop between binding poses to determine the more favorable pose).