Figure : Scheme of the automated absolute binding free energy workflow implemented in the A3Fe python package. a) Following initial system preparation, b) individual 5 ns equilibration runs were carried out for each of the N replicate runs for each leg to provide diverse input conformations for the free energy calculations. c) The first of these for the bound leg was analysed to select Boresch restraints. d) A single short replicate was run for each of the default λ values to provide an estimate of the variance of the gradient, from which the final λ values were calculated. The same Boresch restraints and λ scheme were used for all replicate production runs. e) Simulation time was allocated by iteratively calculating the inter-run standard errors of the per-window ∆G estimates, estimating the optimal run time for each window, and allocating sampling time accordingly. Once the estimated optimal sampling times were achieved, f) the equilibration time was determined for each stage using an ensemble-based equilibration detection heuristic, g) and the overall free energy changes were calculated using MBAR.