The complete workflow (Figure ) was carried out using the python package A3FE, which is available on Github at github.com/michellab/a3fe and is built on BioSimSpace and SOMD. The final 5 ns equilibration run of the fully interacting complex for the first replicate was analysed to select the Boresch restraints using BioSimSpace. This algorithm fits the force constants to the fluctuations observed for prospective sets of anchor points, then selects the stable anchor set which maximally restricts the configurational volume available to the fully decoupled ligand. This effectively mimics strong native receptor-ligand interactions and ensures that ∆G Bound, Restrain ≈ 1.23 kcal mol -1 (see section S6 in Clark et al. for a discussion and Hedges et al. for a tutorial). The same restraints were used for all replicate runs to ensure that the free energy gradients at a given λ value would converge to the same values with infinite sampling.