Due to the coupling between the pseudoparticle and the reaction coordinate, the biasing potential indirectly influences the dynamics of the system. The simulation is considered converged when the pseudoparticle samples a sufficiently uniform distribution. The biasing potential required to achieve this, is then equal to the PMF. Further details of AWH can be found in the original publication, and instructions are available in the official GROMACS documentation. Additionally, GROMACS provides tutorials on the usage of the AWH method, 107,108 in which the application details are highlighted. We also suggest that readers interested in using the AWH method in GROMACS follow the BioExcel webinars, in which both technical and application details of the method were covered. With the AWH method, we computed the dissociation-free energies of barnase-barnstar and benzamidine-trypsin systems. Figure shows the PMF profiles computed from AWH simulations for the barnase-barstar complex (system 1), and the trypsin-benzamidine complex (system 4). While with a single walker (Figure , C) the differences between repeats are smaller than for the US, but still in the order of 5 kcal/mol, this difference reduces to 1-3 kcal/mol when multiple walkers are applied (Figure ). The convergence of the PMF profiles as a function of time for AWH demonstrates a similar trend (Figures ). For single-walker simulations, 400 ns is not sufficient to achieve convergence for both trypsin-benzamidine and barnase-barstar complexes, as the profiles continue to evolve (Figures S34, S36). In contrast, with four walkers the PMF profiles for both trypsin-benzamidine and barnase-barstar complexes, converge within 100 ns (Figures S33, S35), demonstrating the higher efficiency of multiple-walker simulations compared to single-walker ones. Similar trends have been demonstrated before for pulling solutes across membrane channels and for characterizing conformational changes in riboswitches. An additional advantage of the multiple walkers is that the bound state is sampled more extensively as compared to a single walker simulation (Figures and). In the single walker trajectory of the barnase-barnstar complex (Figure ), the bound state is undersampled and the system does not return to the bound state during the simulation, which affects the PMF profile (Figure ). In contrast, we observe that in the multiple walker AHW simulations, at least one of the four walkers samples the bound state extensively (Figures and). Thus, based on previous and our results, we consider adaptive-biasing approaches more robust and reliable than US simulations.