Another possible reason for non-converged ABFE calculations was discussed by Ansari and co-workers, who demonstrated that water networks can play a key role in the binding process of a ligand. In the case of the trypsin-benzamidine system, a water molecule in the binding pocket initiates the ligand release process. In the unbound state, there is a water network replacing the ligand in the binding pocket. However, while also in our simulations, water networks form during the pulling simulations, the structures of these networks differ significantly between the repeats of the US simulations (Figure ). In addition, within a single repeat, the network changes in simulations of the single umbrella windows (Figure ). The average number of water molecules around residues Y228 and D189 fluctuates between the US repeats (Figure ), pointing to a lack of structural convergence in the binding pocket, which, according to Ansari et al., can influence the resulting PMF profiles. While the wetting/dewetting transition of the binding pocket often plays a key role in ligand binding, it is not the only determinant defining the protein-ligand and proteinprotein dissociation. Our results, unfortunately, demonstrate that for large biomolecular systems like protein-protein or protein-ligand complexes, a straightforward application of the umbrella sampling protocol for a single reaction coordinate, as widely used by the community, may not always provide reliable binding free energies by itself. Similar results were also obtained for solute permeation across membrane channels, for which solute pulling in two directions led to major hysteresis effects. As a conclusion, despite starting the pulling from the same equilibrated structure and applying orthogonal restraints, each pulling simulation results in a slightly different pathway along the reaction coordinate. Such differences in the pathways, even if small, can result in differences of at least a few, or even tens of, kcal/mol in the resulting PMF. Therefore, a single set of US using a simple distance-based reaction coordinate is not sufficient for obtaining a reliable estimate for protein-protein, protein-peptide, or protein-ligand affinities.