These are restrained to prevent consequential interactions, and then the work required to alchemically transform (i.e., A H p → A - p and A - s → A H s ) these residues into their complement (i.e., (de)protonated form) is computed. This construct ensures a neutral simulation box at all times during an alchemical transition. Given two equilibrium ensembles (e.g., A p and A s ), the distributions of work values generated by rapidly transforming residues from the first ensemble into residues from the second (and vice versa) allow one to estimate the free energy difference. Here, the transformation is alchemical, the transitions are on the order of 100 ps, and the free energy difference is estimated using Bennett's acceptance ratio (BAR) relying on the Crooks fluctuation theorem 60 (CFT). Previous work has demonstrated the ability of this NEQ approach to resolve folding free energies and binding affinities within experimental uncertainty.