The non-equilibrium simulations, each 500 ps long, were run from each conformation to the opposite coupling state of the ligand by linearly interpolating the Hamiltonian between the two end-states. The gradients ∂H(λ, x)/∂λ were integrated over the course of each nonequilibrium simulation to obtain the amount of work performed. Free energies were computed from the work distributions in both directions using a maximum likelihood estimator based on the Crooks Fluctuation Theorem 11 by means of pmx . Finally, free energy estimates from different legs of the thermodynamic cycle were combined and the contribution of restraining the decoupled ligand to the protein was added 7 incorporating the correction for the correlations in the restrained degrees of freedom (Section 2).