We also investigated whether the use of enhanced sampling improved the convergence and efficiency of the simulations. We compared three protocols, one without enhanced sampling, another using HREX and a third protocol, ϵ-HREX, where we scale down the LJ-ϵ by a factor of γ = 0.5. Scaling down the LJ-ϵ can soften interactions and lower energy barriers between conformational states, and can therefore, coupled with replica exchange swaps, improve sampling. All three protocols converged to statistically the same relative binding free energies (Figure ). The standard deviation, calculated across three independent repeats, was highest when no enhanced sampling was used, while the SepTop HREX and SepTop ϵ-HREX protocols had similar values for the standard deviation. Here, we were primarily focusing on comparing results from different methods with one another rather than comparing to experimental binding affinities, because methodological improvements do not always improve agreement with experiment. In particular, agreement with experiment is not just a function of sampling, but of multiple factors, such as the force field and the choice of protonation states, counterions, etc., meaning that the best method may not necessarily agree best with experiment except when all other issues have been addressed.