Moreover, we computed the RMSD of the ligands between the initial docked poses and the final states at the end of the MD simulations for the two protonation states of 4EWO: i) Asp32 H , Asp228 H and ii) Asp32 H , Asp228 -. We used Chimera to align the active site of each initial BACE-1-ligand complex to the last frame of the corresponding MD trajectory. The alignment of the active site was done within 5 Å of the ligand. Then, we computed the RMSD values with Chimera. Out of 75 ligands, 54 had lower RMSD values when docked and simulated in BACE-1 with the single protonated aspartyl dyad (Asp32 H , Asp228 -). The RMSD values are reported in the Supplementary Information available on MMGBSA/blob/master/RMSD.csv. This result shows that the binding mode of BACE-1 ligands depends on the protonation states of aspartates 32 and 228 and confirms that it is better to model the protonation state of BACE-1 as Asp32 H , Asp228 -for the given ligand dataset. Overall, these findings suggest that in large-scale binding affinity calculations, it is better to use the protonation state of the apo form of the protein.