All simulations were carried out with the GROMACS 2019.4 molecular dynamics engine 1 modified to correctly handle pair interactions within a decoupled molecule larger than the electrostatic cutoff (bug 3403). The Am-ber99sb*ILDN force field was used for the proteins throughout this work together with the TIP3P water model. Ligand parameters were taken from the previous relative free energy study parametrized with the General Amber Force Field (GAFF v2.1) 49 using AM1-BCC charges assigned with ACPYPE and AnteChamber . Initial ligand binding poses were reused from a previous relative binding free energy study , where the ligands were modeled based on similar compounds in existing holo crystal structures (Table ). Initial protein structures were stripped of surrounding water (if present) and resolvated in dodecahedral simulation boxes with 1.5 nm of padding between solute and box edges. For apo simulations started from holo structures the ligands were removed before adding water, so that the binding cavities could also be filled with water. Effects of retaining crystallographic water before filling the remaining cavities were also examined (Fig ). Ions were added to neutralize the system and reach a salt concentration of 150 mM.