All MD simulations were performed using GROMACS 2018.3. In the EQ approach, every λ window was energy minimized using steepest descent for 5000 steps, then equilibrated in the canonical ensemble for 10 ps at 298.15 K. Afterwards, the system was equilibrated in the isothermal-isobaric ensemble for 100 ps at a pressure of 1 bar. The production run was performed at constant NPT as well. All MD simulations were performed using the leap-frog integrator at a timestep of 2 fs. In the alchemical pathway, first the electrostatic interactions were decoupled, then the sterics, applying a soft-core potential. The soft-core potential is used to avoid instabilities in intermediate lambda windows. Although new softcore potential functions, including the smoothstep softcore potentials, have been developed recently, the soft-core potential from Beutler et al. was used in this work as this is widely available in GROMACS. EQ and NEQ methods were treated equivalently with respect to the soft-core potential, thus both approaches were evaluated at equivalent conditions. The intramolecular interactions of the ligand were not changed during the process. Complete settings can be found in the mdp files provided in the SI. Although the convergence rate can be different among λ windows, we ran all alchemical states for an equal amount of time for convenience. The overall convergence of the free energy difference will therefore be determined by the slowest converging window.