On the other hand, for the EQ approach, the final ∆G estimate depended on the equilibration time (how much data were discarded to equilibration). Discarding more data (20 ns per λ window) made a difference of ∼ 1 kcal/mol in all three replicates compared to not discarding any data to equilibration (SI Figure ). This and a low number of uncorrelated samples in some λ windows can indicate a slow degree of Figure . Free energy difference of decoupling the HSP90 ligand in the binding site as a function of total simulation time. For the EQ approach we show the mean free energy difference across three replicates while for the NEQ approach the free energy estimate was obtained by pooling work values from three replicates and estimating one free energy difference using BAR (Sec. 4.5). The uncertainty estimate in both approaches is the standard deviation across three replicates. The three NEQ protocols differ in the length and number of the switching transitions. A high standard deviation indicates inadequate sampling. freedom (DOF). Similar to the T4 lysozyme system, analyzing sudden changes in dH/dλ values helped identifying these slow DOF, as shown below.