Alternatively, a so called path-based approach may be adopted to compute proteinprotein binding free energies with MD methods. In this approach, the potential of mean force (PMF) for dissociating a protein assembly can be computed via a variety of enhanced sampling techniques applied to pre-selected Collective Variables (CVs). Whilst the pathbased PMF approach appears more tractable, it is still difficult to obtain converged free energy estimates for large biomolecules with complex geometries. More precise free energy changes can be obtained through the application of restraints which ensure that the relative orientation and position of the ligand with respect to the receptor is preserved and therefore the available phase space to be sampled during the simulations is reduced. To this end, Woo and Roux pioneered the so-called geometrical route for binding free energy calculations, where the binding process is decomposed into distinct, independent processes, each described by a unique CV, which corresponds to a specific slow degree of freedom of the system. In the original implementation of the geometrical route, three atom centres are chosen on both binding counterparts, constructing a local frame of reference. The orientation of the ligand with respect to the receptor is then defined by the three Euler angles Θ, Φ, Ψ, and the position of the ligand with respect to the receptor is defined by the polar angles θ and ϕ. The ligand is additionally restrained in the average conformation it adopts when bound to its receptor with the application of an RMSD restraint. The binding free energy is then quantified by means of a series of sequential PMF calculations on the above CVs. In each PMF calculation, the only restraints present are those that have been added beforehand. The final PMF calculation involves the physical separation of the two binding partners along the Euclidean distance r between their centres of mass and is carried out in the presence of all restraints. Simulations without such restraints have been reported to fail to converge and to yield binding free energies inconsistent with experimental data. The geometrical route has been automated with quaternion-based angle restraints as CVs in the NAMD and GROMACS 72 MD engines. Additional variations of the framework have also been introduced, with various implementations of RMSD restraints applied on both receptor and ligand in the bound and unbound states. The protocol has so far been successfully used in calculating the binding free energy of a variety of protein-protein complexes, in good agreement with experimental data, including a homodimeric α-helical complex. An alternative set of orientational and positional restraints has been proposed by Boresch et al. in the context of small molecule-protein binding free energy calculations. Boresch restraints are constructed by selecting three anchor points (atoms or groups of atoms) on the receptor and three on the ligand. The restraints correspond to the external degrees of freedom of the ligand, and consist of a distance r, two angles θ A , θ B and three dihedrals ϕ A , ϕ B , ϕ C . The Boresch angles and dihedrals play the same role as the Euler and polar angles used in the geometrical route, but are easier to implement in other MD software, such as OpenMM. Numerous techniques having been developed to facilitate the automated selection of anchor points. To the best of our knowledge, Boresch restraints have not been used so far in binding free energy calculations via a geometrical route. We decided to employ the Boresch angles and dihedrals instead of the Euler and polar angles, since no quaternion implementation is available in OpenMM to date. In principle, the binding free energy should be independent of the type of restraints, the atom centres selected for the construction of the restraints, or the values of the force constants, provided stable restraints that have been appropriately selected. In the present work, we combine Well-Tempered Metadynamics (WT-metaD) with Boresch restraints 77 in OpenMM to explore the oligomerisation and topology preferences of de novo designed coiled coils. The overall pipeline is summarised in Figure . We demonstrate the applicability of our protocol by predicting: the topology (parallel vs antiparallel) preferences of CC-Di, CC-Tri and apCC-Di, the oligomerisation state preferences of CC-Di, CC-Tri and CC-Tet, and the pH switching behaviour of the CC-Hex*-L24E system, which forms a parallel hexamer at low pH and an antiparallel tetramer at high pH (Figure ). We also show that employing WT-metaD and funnel metadynamics 90 in the absence of orientational and positional restraints fails to yield reliable free energy estimates. Overall our results demonstrate the potential of path-based MD methods to act as a late stage filter in multi-state protein design.