This can be partly attributed to the increased number of available conformations of the separated coiled-coil monomers in the bulk solvent. We therefore investigated whether funnel metadynamics could yield better reproducibility via restricting the bulk space available to the monomers upon dissociation. Whilst improved reproducibility was achieved in certain cases, this was found to be largely dependent on the shape of the funnel, and differed between coiled-coil assemblies (Figure ). Inspection of the bound states from the simulations revealed further complications. Whilst sometimes reasonable bound states could be obtained (Figure ), in the majority of simulations unreasonable bound conformations prevailed (Figure ). Analysis of inter-helical contacts from the bound state conformations showed a wide variety of contacts formed, which are not present in the native coiled-coil dimers (Figures ). Interestingly, hotspots of interactions appear approximately every seven residues, which can be explained by the repetitive nature of coiled coils. The monomer sequences of CC-Di and apCC-Di consist of 4 heptad repeats each; it is plausible that when the monomers attempt to re-associate, heptad i of one helix can form interactions with heptad i-1 or i+1 from the other helix, resulting in the partial stabilisation of misfolded structures (Figure ). This is further exacerbated by the formation of non-native salt bridges, a common caveat of implicit solvent. For example, Glu2 in helix A in CC-Di forms non-native salt bridges with Lys14 and Lys21 from helix B, whereas the native salt bridge with Lys7 is not formed (Figure ). Similar salt bridge traps can be seen in apCC-Di, e.g. Glu4-Arg20, Glu4-Arg21 and Glu6-Arg21 (Figure ). This renders the obtained PMF profiles unreliable, as the minima do not correspond to the desired bound states. In fact, multiple local minima can be observed, corresponding to different misfolded structures with non-native contacts (Figure ).