2 ) for the bond and 20 kcal/(mol * rad 2 ) for angles and dihedrals. A table of the protein and ligand atoms used to restrain the complex can be found in SI Table . The atoms were selected during the setup of the system with YANK. YANK's selection process picked heavy atoms that are not co-linear and ensured that the distance restraint involved a protein-ligand atom pair within 1-4 Å. Previously, the calculated binding free energy was shown to be independent of the atom selection and force constants. Here, the restraint contribution (Figure panel A) was calculated by running two equilibrium states, one of the coupled, restrained ligand and one of the coupled ligand without restraints for 10 ns each. The free energy difference was estimated using BAR and three independent replicates were run to check for convergence and to estimate the uncertainty.