The last 20 ns of our equilibration procedure was analysed using the Nwat-MM/GBSA method . Here the N = 20 nearest waters to the ligand in the binding site are included as part of the protein in the MM/GBSA calculation. GROMACS XTC files were converted to AMBER NETCDF format using MDAnalysis 1.1.1 , and the trajectory and topologies manipulated to remove excess waters using cpptraj v5.1.0 . The mmbondi2 radii with GB model 2 parameters were employed with a GB salt concentration of 150 mM. Frames were sampled every 25 ps from the 20 ns trajectories. The AmberTools21 versions of MMPBSA.py and sander were used for the MM/GBSA calculations 77 .