In Figure , we show the computed PMF profiles for the dissociation of the five systems described in the Methods section and illustrated in Figure using the US procedure. For all simulated systems, the PMF profiles differ for individual repeats. Differences in the PMF depth (∆W R ) between repeats lie in the range of ∼2-20 kcal/mol (Table ), which is much higher than the PMF errors calculated with the bootstrapping method usually employed for the WHAM, which is in the order of only 0.5-2 kcal/mol. Large differences in the ∆W R between repeats can be problematic because the binding free energy between different ligands to a binding site may vary by about the same amount, making it difficult to differentiate between ligands with US. To rule out potential effects of the force field or software, we additionally calculated the dissociation PMFs for the clpS protease adaptor complexed with LLL tripeptide (system 3) using Amber99sb-ildn FF with GROMACS and CHARMM36 with OpenMM 69 software. The differences between the US repeats were also observed for those simulations (Figure ).