Prior to production runs with US or AWH, the potential energy of systems 1-4 was minimized using the steepest descent algorithm, followed by equilibration of 50 ps in NVT and 50 ps in NPT ensembles. During the NVT and NPT equilibration, position restraints of 1000 kJ mol -1 nm -2 were applied in all directions on the C α atoms of the proteins and peptide, and on all heavy atoms of the ligand in system 4. During pulling, one of the protein chains was restrained and the other chain (either protein, peptide, or lipid) was pulled away from it. We refer to the restrained protein chain as a fixed one, and to the one which was pulled away as the mobile one. During pulling, US and AWH simulations of systems 1-4 the following restraints were applied: 1000 kJ/mol/nm 2 position restraints in all directions for the C α atoms of the fixed protein chain, and orthogonal position restraints in x-,ydirections with a force constant of 1000 kJ/mol/nm 2 for the C α atoms of the mobile protein or peptide, or heavy atoms of the ligand. For system 5, following the procedure of Lemkul and Bevan, position restraints of 1000 kJ/mol/nm 2 in all directions were only applied to the C α atoms of the subunit next to the one dissociated from the aggregate. To test the effect of position restraints in x,y-directions, we also performed additional simulations for the trypsin-benzamidine (system 4) using different force constants k xy = 4184 kJ/mol/nm 2 and k xy = 0 kJ/mol/nm 2 Pulling and umbrella sampling. As the aim was to perform multiple repeats for umbrella sampling (Figure ) and compare the PMF profiles, we first performed multiple repeats of the non-equilibrium pulling to get initial structures for umbrella windows along the dissociation coordinate. Starting from the equilibrated system, the mobile protein, peptide, or ligand was pulled away from the fixed protein along the z-axis. Multiple repeats (5 repeats for systems 1-4 and 10 repeats for system 5) of pulling for each system were performed, all starting from the same initial structure but with different initial velocities.