  We introduce the self-Relative Binding Free Energy (self-RBFE) approach to
evaluate the intrinsic statistical variance of dual-topology alchemical binding
free energy estimators. The self-RBFE is the relative binding free energy
between a ligand and a copy of the same ligand, and its true value is zero.
Nevertheless, because the two copies of the ligand move independently, the
self-RBFE value produced by a finite-length simulation fluctuates and can be
used to measure the variance of the model. The results of this validation
provide evidence that a significant fraction of the errors observed in
benchmark studies reflect the statistical fluctuations of unconverged estimates
rather than the models' accuracy. Furthermore, we find that ligand
reorganization is a significant contributing factor to the statistical variance
of binding free energy estimates and that metadynamics-accelerated
conformational sampling of torsional degrees of freedom of the ligand can
drastically reduce the time to convergence.
