Energy minimization Once the placement of all water layers is completed, the system is minimized using OpenMM . Protein heavy atoms are restrained with a 2.5 kcal/mol/Å 2 force constant, while water molecules are free to move. The minimization is done with a non-bonded cutoff of 9 Å and no periodic condition. The following minimization steps were tested: 50, 75, 100, 125.