Simulations of the competing up-down-up variant of CC-Tri served two purposes: to assess whether the correct trimer topology can be predicted, and to verify that the order by which the helices are separated from the assembly does not affect the total binding free energy of the complex. While the individual free energies from each step are expected to be different depending on the helix being removed, the total binding free energy should be the same, given that free energy is a state function. To this end, the up-down-up trimer was separated with two different schemes (Figure ). The total binding free energies from the two schemes were consistent with each other (Figure , Tables ), demonstrating that the protocol does not depend on the order in which the monomers are removed from the complex. Additionally, the up-down-up CC-Tri is predicted to be less favourable than the all parallel CC-Tri, establishing that our framework is able to predict the preferred topology of a higher order coiled coil. Interestingly, the ∆G • bind of the dimer step from the parallel CC-Tri simulations (-16.2 ± 1.2 kcal mol -1 , Table ) is consistent, within error, with the ∆G • bind of the dimer step from the up-down-up CC-Tri simulations with scheme 2 (-15.8 ± 2.0 kcal mol -1 , Table ), which is in agreement with the fact that these two intermediate