Standard harmonic potentials, which act only outside the colvar range, are applied to keep the system inside the windows during the equilibration and production runs. These restraints act like walls against which the groups related to the ξ definition will bounce. A force constant of at least 80 kcal mol -1 Å-1 is used to impede accumulation of samples near the borders, which would compromise the homogeneous inspection of phase space. The second strategy consists of using an importance sampling approach in order to sample all ξ values with an equal probability regardless of the ruggedness of the potential energy landscape. The ABF methodology was chosen given its conceptual and practical simplicity. For instance, consecutive windows do not need to overlap, as in umbrella sampling or metadynamics, with outstanding savings in simulation time. In essence, ABF computes the mean thermodynamic force along the collective variable, which is then cancelled out by an equal and opposite biasing force. The inclusion of this biasing force allows the system to move seamlessly along the colvar as if the process was governed by diffusion, overcoming the metastable regions and recovering ergodicity. Theoretically, ξ is a continuous variable, but in practice the collective variable is divided into small bins of width δξ, where samples are accrued into each bin to compute the mean force in the interval (ξ 0 , ξ 0 + δξ). In all of our simulations, the bin width δξ is 0.5 Å, which permits to capture the fine details of the continuous PMF without compromising the number of force samples accumulated in each bin. As the simulation progresses, more samples are accrued within each bin and so the estimation of the mean force from the instantaneous thermodynamic forces F gets refined towards its actual value. The thermodynamic integration formalism, on which ABF is based, allows to yield the PMF as the mean force along the collective variable is simply the negative gradient of the free energy: