Impact of Varying Velocities and Solvation Boxes on Alchemical Free-Energy Simulations.

Alchemical free-energy perturbation (FEP) is an accurate and thermodynamically stringent way to estimate relative energies for the binding of small ligands to biological macromolecules. It has repeatedly been pointed out that a single simulation normally stays near the starting point in phase space and therefore underestimates the uncertainty of the results. Therefore, it is better to run an ensemble of independent simulations. Traditionally, such an ensemble has been generated by using different starting velocities. We argue that it is better to use also other random choices made during the setup of the simulations, in particular the solvation of the solute. We show here that such solvent-induced independent simulations (SIS) sometimes give a larger standard deviation and slightly different results for the binding of 42 ligands to five different proteins, viz. human N-terminal bromodomain 4, the Leu99Ala mutant of T4 lysozyme, dihydrofolate reductase, blood-clotting factor Xa, and ferritin. SIS does not involve any increase in the time consumption. Therefore, we strongly recommend the use of SIS (in addition to different velocities) to start independent simulations. Other random or uncertain choices in the setup of the simulated systems, e.g., the selection of residues with alternative conformations or positions of added protons, may also be used to enhance the variation in independent simulations.