. Free energy difference for decoupling the HSP90 ligand in the binding site. We show EQ and NEQ protocols with and without the use of HREX as an enhanced sampling technique. In the NEQ HREX approach, end state structures were used from the EQ HREX simulation, and no enhanced sampling was performed in the NEQ switches. 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. Using structures from HREX simulations as input for the NEQ switches did not improve results in this case. On the contrary, additional sampling problems were introduced (see Figure ).