The errors in our ABFE estimates were within 0.8 kcal mol -1 (Table ). Except for one HIV-1 Protease system (1EC0) and the BPTI system (5PTI), all other systems resulted in errors within 0.5 kcal mol -1 . For all the systems, the application and removal of the binding site restraints resulted in the largest errors in our calculations, but each of these errors was generally below 0.4 kcal mol -1 . This relatively large statistical error could be expected because constraining the protein is a relatively difficult task. To constrain the orientation of the binding site to its orientation in an equilibrated starting structure of the simulation, we applied position restraints to a significant number of heavy atoms of the binding site, i.e., in the middle of the protein (see Section 2.3.3 for details). Across the three simulation replicates, variations in motion of the protein can result in different strains on the restrained heavy atoms, and hence, the free energy to restrain the binding site can exhibit relatively large statistical error.