Sampling Challenges of MM/PBSA Binding Energy Calculations

The accuracy of the MM/PBSA binding free energy calculation depends on both the employed force field and the statistical quality of sampling. However, the impact of sampling sufficiency has often been underestimated in previous studies. Here, we systematically analyze multiple protein-ligand systems using conventional and enhanced molecular dynamics simulations of different lengths. Our results show that short simulations may sometimes give the illusion of convergence while failing to capture slow conformational transitions that affect the computed free energies. Longer or enhanced simulations can reveal these hidden motions but do not always improve agreement with experiments, indicating that force-field limitations may dominate once statistical convergence is achieved. From a theoretical perspective, sufficient sampling is the fundamental requirement for converging thermodynamic quantities such as ΔΔ<i>G</i>, independent of the simulation length. Practically, the degree of sampling sufficiency depends on system-specific dynamics and research goals. This work highlights the importance of recognizing sampling sufficiency as a statistical prerequisite rather than equating it with long simulations and calls for more adaptive strategies to balance efficiency and reliability in MM/PBSA analyses.