Instead, following You et al., we attribute the discrepancies between the PMF profiles of the individual repeats to differences in the pathways along the reaction coordinate. In the US method, those pathways are defined by the reference configurations for the harmonic restraints in each US window. For the HdeA dimer (system 2) and the amyloid β-peptide A β 42 (system 5) differences in pathways were observed for different repeats, as shown in Table : Estimated PMF depths ∆W R values for the repeats of the US (systems 1-5) and the multi-walker AWH (systems 1 and 4) simulations. ∆W R was calculated from the PMF profiles presented in Figure as the mean of the flat region of the PMF (cut-offs of flat region 3.0, 2.75, 1.7, 1.9 and 3.5 nm for US systems 1-5, respectively, and 3.0 and 1.2 for AWH systems 1 and 4, respectively.) (RMSDs) between reference structures for the umbrella windows of different repeats were around 0.1 nm, indicating that the pathways were very similar (Figure ). Moreover, exchanging the reference structures between repeats while keeping the initial coordinates the same, reveals a strong dependency of the PMF on those reference structures (Figure ). Since the sequence of reference structures defines the pathway for the US, these results