To calculate the free energy difference between the end states along the path defined by the intermediate λ states, we used the Multistate Bennett Acceptance (MBAR) method as the equilibrium free energy estimator. MBAR relies on equilibrium sampling of a single path between the end states through the intermediate λ states. For a reliable free energy estimate through MBAR, the simulations of the λ states need to be long enough (generally in the order of tens to hundreds of nanoseconds (ns)) to generate a representative sampling of the Hamiltonian, though exact time requirements depend on the characteristic timescales of the system. 2.1.2. Non-equilibrium switching (NES) free energy estimate. The NES free energy estimate relies on many independent and short simulations ('switches') that bridge the end states by transitioning between them (out of equilibrium) in both directions. The Hamiltonian of the system is coupled to a parameter λ and is rapidly changed (usually within a few hundred picoseconds (ps) or less) from one end state to the other. The NES switches are started from configurations drawn from a representative equilibrium sampling of each of the end states. The NES switches result in a collection of sampled paths between the two end states. Because of the non-equilibrium nature of the short switches, there is work dissipation along the path of each of the switches, which needs to be accounted for.