The obtained binding free energies ∆G • bind,total /helix are in agreement with the experi-mental structures, namely CC-Di preferably forms a dimer, CC-Tri preferably forms a trimer and CC-Tet preferably forms a tetramer (Figure , Tables S2, S5, S8-S14). Similar to the original geometrical route framework, the largest contributions to the binding free energy stem from the separation and RMSD PMFs, with their uncertainties also contributing the most to the reported standard error. Examination of the ∆G • bind of each intermediate state (Figure ) reveals which helix dissociation event contributes the most to the total binding free energy of the complex. In the case of CC-Di, the dimer dissociation event contributes the most when CC-Di is modelled as a trimer and tetramer. Additionally, it can be seen that the binding free energy for the native dimeric CC-Di is more favourable compared to the binding free energy for the intermediate dimer states in the trimeric and tetrameric CC-Di complexes (Tables S3, S8, S9). These in-termediate dimer states have been restrained in their 'open' conformations and therefore lack multiple intermolecular interactions present in the native dimeric CC-Di, which renders their constituting monomers more prone to dissociation. In the case of CC-Tri, the trimer dissociation step contributes the most when CC-Tri is modelled as a trimer and tetramer. In the case of CC-Tet, the trimer dissociation step has the highest contribution. The breakdown of the contributions of each PMF to the binding free energy (Table ) reveals that while both separation free energies in the tetramer and trimer steps are negative, it is the contribution of the RMSD PMFs in the bound and unbound states ∆G RM SD bound and ∆G RM SD unbound that distinguishes the two dissociation steps. By definition ∆G RM SD bound <0 and ∆G RM SD unbound >0, so the term ∆G RM SD bound + ∆G RM SD unbound quantifies the loss of internal conformational freedom. This term is highly positive in the case of the tetramer, but less pronounced in the case of the trimer. This overall difference makes the trimer dissociation step appear more favourable. Nevertheless, CC-Tet is still predicted to preferably form a tetramer.