Given a set of initial docked poses, the precision of our ABFE calculations is affected by two levels of numerical uncertainty. One, the numerical convergence of the free energy calculations as a function of simulation time, is estimated by blocking analysis to be about 0.5 to 3 kcal/mol (Table and SI Tables). This uncertainty could be reduced by using longer MD runs. The other source of uncertainty is evident from the differences between the two independent ABFE runs started from the same set of docked poses, one of which yielded the more favorable BFEs considered in the prior subsections, the other of which yielded the less favorable BFE as shown in Table and SI tables. The differences between these two runs range from 0 to about 9 kcal/mol. Detailed examination of cases where this deviation is large reveal that they occur when the MD equilibration stages of the two independent runs, which are initiated from the same docked pose, lead to significantly different post-equilibration conformations. The ligand conformational restraints routinely applied at the next stage of the ABFE calculation can lock in this conformational difference, leading to significantly different ABFE results. For example, the same initial ligand pose from docking of the BACE1 ligand CHEMBL1090542 relaxes to two quite different conformations in the two independent MD equilibration phases. In one run, the ligand conformation stays close to the initial pose from docking (Fig , left and middle panels), resulting in the more favorable ABFE of -16 kcal/mol. In the other run, the ligand loses its initial close interaction with the catalytic Asp residues (Fig 3 right panel), resulting in the less favorable binding free energy of -6 kcal/mol. Note that similar problems can occur if the receptor makes a conformational transition that equilibrates slowly relative to the simulation time.