We calculated the PMF profiles of the dissociation for the following systems (Figure ): (1) barnase-barstar (PDB ID: 1BRS 72 ) (2) HdeA dimer (PDB ID: 1BG8 73 ) (3) clpS protease adaptor with LLL tripeptide (PDB ID: 3G19 74 ) (4) trypsin-benzamidine (PDB ID: 3PTB ) and ( ) amyloid β-peptide Aβ 42 (PDB ID: 2BEG ). For all systems, we performed multiple repeats of US simulations. The AWH simulations were performed for systems 1 and 4. For systems 1-4, the interactions were modeled using the CHARMM36 all-atom force field (FF). Benzamidine in system 4 was parameterized with CGenFF. For system 5, the GROMOS96 53A6 parameter set was used, as in the earlier work of Lemkul et al. Additionally, to rule out bias of the force field, we repeated the simulations of system 3 using the Amber ff99SB-ILDN FF. We also ran system 3 with CHARMM36 force field but with OpenMM software to check that discrepancies between repeats are not softwarespecific. Systems 1-4 were constructed using the following procedure: (i) The structure for each protein system 1-4 was obtained from the Protein Data Bank 80 and placed in a periodic rectangular box; (ii) The C-and N-termini were kept charged, and the protonation states for the titratable residues were selected for pH=7: all Glu, Asp and His deprotonated, and Lys protonated; (iii) The proteins were oriented in the simulation box such that the reaction coordinate of the pulling simulation was aligned with the z-coordinate, as in the Figure : Structures of the simulated systems, for which the PMF of the dissociation was calculated using umbrella sampling. (1) barnase-barstar 1BRS (2) HdeA dimer 1BG8 (3) clpS protease adaptor with LLL tripeptide 3G19 74 (4) trypsin-benzamidine 3PTB 75 and (5) amyloid β-peptide Aβ 42 2BEG. The protein, peptide, or ligand dissociated from the protein is shown in orange. earlier work, and the reaction coordinate was defined either as the vector connecting the center-of-masses (COMs) of the two proteins or protein and peptide (systems 1-3), or as the vector connecting the COM of heavy atoms of the ligand and the C γ atom of Asp-189 (system 4) as was proposed in previous work; (iv) Systems were then solvated with CHARMM TIP3P or TIP3P water molecules. Systems were neutralized by adding Na+ and Clions at 0.15 M concentration. For system 5, the initial structure was an equilibrated snapshot from the tutorial of Lemkul, 84 based on their earlier publication. The sizes of the simulation boxes of our systems were: barnase barstar 9x9x15 nm 3 box with ∼38000 waters, HdeA dimer 8x8x15 nm 3 with ∼31000 waters, clpS protease adaptor 6x6x12 nm 3 with ∼13500 waters, and trypsin-benzamidine 9x9x12 nm 3 with ∼31000 waters. System 5, the amyloid β-peptide, comprised a box of dimensions 11x10x12 nm 3 with ∼42000 SPC 85 waters. All input configurations, together with topology and run parameters, are provided as Supporting Information.