Notably, this was not possible in the funnel metadynamics simulations in the absence of orientational and positional restraints. We carried out two sets of simulations for CC-Tri, one with and one without RMSD restraints on the receptor helices (Tables S4-S5, Figures ). This served as a control for the additional RMSD restraints, as the binding free energy should be the same after correcting for the presence of restraints, regardless of the type and number of restraints used. When the contribution of the RMSD restraints in the bound and unbound states is accounted for, the binding free energy for the trimer step is consistent with that from the simulations without the RMSD restraints (Figure , Table ).