On Restraints in End-Point Protein–Ligand Binding Free Energy Calculations

The impact of harmonic restraints on protein heavy atoms and ligand atoms on end-point free energy calculations is systematically characterized for 54 protein–ligand complexes. We observe that stronger restraints reduce the equilibration time and statistical inefficiency, suppress conformational sampling, influence correlation with experiment, and monotonically decrease the estimated loss of entropy upon binding, leading to stronger estimated binding free energies in most systems. A statistical estimator that reweights for the biasing potential and includes data prior to the estimated equilibration time has the highest correlation with experiment. A spring constant of 20 cal mol⁻¹ Å⁻² maintains a near-native energy landscape and suppresses artifactual energy minima while minimally limiting thermal fluctuations about the crystal structure. © 2019 Wiley Periodicals, Inc.