For MM-PBSA calculation the 50-ns MD production trajectories were reduced to 501 frames each, by extracting a frame every 100 ps. The resulting snapshots of the MD simulations were utilized for post-processing free energies by the single-trajectory MM-PBSA method implemented in g mmpbsa. Six different dielectric constants ( = ) were used for the binding pocket, while the solution dielectric constant was kept constant at s=80. Calculations are performed based on a homogeneous medium with a range of dielectric constants for the solute, an ionic strength of 153.6 mM, an ionic radius of 0.95 Å for positive charged ions and 1.81 Å for negative charged ions, and a solvent probe radius of 1.4 Å. An example configuration file for g mmpbsa is provided within the supplements (Listing 1). Other parameters influencing the grid dimensions of the calculation, such as 'cfac', 'gridspace' and 'fadd' were varied from suggested defaults (1.5, 0.5 and 10, respectively) showing only marginal variations in the results and therefore not further changed.