the syn-anti isomerisation in MIF180 was rarely sampled, in contrast to our previous work using GAFF2.11, 103,118 and we performed calculations for the charged ligand PIP2 using a reaction field treatment of electrostatics without maintaining neutrality of the box or applying corrections. 120 Our main interest was in the equilibration and convergence behaviour of the non-adaptive runs, and their relative performance compared to adaptive protocols. However, we include experimental binding free energies in Table to check that our results are reasonable. The 30 ns computed free energies are broadly similar to the experimental values, giving some assurance that our default protocol is reasonable. We note that the calculated values appear excessively negative for ligands larger than benzene, in particular the MDM2 ligands. This is to be anticipated for a wide variety of sampling challenges in equilibrium ABFE calculations, because the bound leg simulations are started from the holo structure. Any failure to relax towards the apo structure and free ligand conformations during the simulation can be regarded as erroneous "preorganisation" of the system for binding, producing erroneously favourable estimates of binding. This effect can generally be expected to increase for larger ligands with more substantial sampling challenges and more favourable binding affinities, and may be avoided using bidirectional non-equilibrium switching protocols which account for the apo state. While we make broad comparisons with experimental affinities here, we note that these are from different sources, including biochemical assays. More pertinently, the initial test systems show a range of equilibration behaviours. While the results obtained for the brief 0.2 ns runs are not significantly different to those from the costly 30 ns runs for T4L, there are significant and substantial differences for MDM2-PIP2, MIF, and especially PDE2a. As shown by the breakdown of binding energy components (Table ) and the convergence plots (Figure ), the dominant contribution to slow equilibration comes from the bound vanish legs, where sampling is impeded by the protein and where a second-order phase-change like transition occurs as the Van der Waals interactions are removed. The diversity of equilibration behaviour suggests that equal-time equilibration rules are likely to be sub-optimal. However, all cases of slow equilibration are similar in that the overall free energies become less favourable with increasing sampling time, which is to be expected given the discussion of sampling issues above.