Multiple distance restraints provide a framework which is free from the inherent instabilities of Boresch restraints, and which allows more complete restraint of the ligand than Boresch restraints alone. However, their naïve application renders the ABFE framework theoretically inexact. To illustrate this, harmonic distance restraints were applied to every heavy atom in the ligand and their lowest variance unique partner heavy atom in the protein (protocol M-All, 22 receptor-ligand distance restraints). As expected, this produced an excessively negative ∆G o Bound estimate, in excess of 3 kcal mol -1 more negative than most of the Boresch restraints, indicating that ∆G Preorg. was large (Table ). The distance restraint dictionaries used for all protocols are given in Section S14. a Results for binding pose A, in kcal mol -1 . Uncertainties stated as 95 % confidence intervals based on the variance of 5 replicate runs assuming Gaussian distributions. The distance restraint dictionaries used for all protocols are given in Section S14. -N indicates that the protocol was repeated with no intramolecular rigidification, -R indicates repetition with release to the single strongest distance restraint, and -1 indicates repetition with the flat-bottomed diameter set to 1 Å for all restraints. ∆G To Dist. Rest. is the free energy of releasing the multiple distance restraints to a single distance restraint.* 10 replicate runs were used. The convergence of -∆G o Release with respect to the standardstatecorrection parameters was confirmed (Section S15). This was contrasted with the rigorous multiple distance restraints scheme with intramolecular rigidification of anchor points (Figure ), referred to as M-Rig. Anchor points were selected as for M-All, except that only anchor points in the phenol moiety of the ligand were used in order to avoid the tight restraints restricting rotatable bonds in the ligand; this would likely require an enhanced sampling approach, such as umbrella sampling, to restrain bond rotation before applying the restrictive intermolecular restraints. This resulted in 7 receptor-ligand distance restraints. The intramolecular restraints were implemented as harmonic distance restraints between all pairs of anchor atoms within the given molecule, with force constants of 75 kcal mol -1 Å-2 . For simulations where -∆G o