Reference calculations were done using an alchemical free energy calculation toolkit known as YANK . YANK provides several schemes for sampling from multiple thermodynamic states. For reference calculations we applied the replica exchange sampler (also known as Hamiltonian Exchange) , using the OpenMM simulation engine . Free energies are estimated using the multistate Bennet acceptance-ratio (MBAR) . (For details on the thermodynamic cycle used in YANK and the theory see )