The entire system was then solvated in a 23.5Å spherical droplet of TIP3P water molecules, centered on the CG atom of D229, and subject to surface-constrained all-atom solvent (SCAAS) boundary conditions. The system was modeled using a multi-layer approach standard to such simulations in which all atoms within the inner 85% of the water droplet are allowed to move freely, the atoms in the external 15% of the droplet are restrained to their crystallographic positions using a 10 kcal•mol -1 •Å -2 harmonic positional restrained, and all atoms outside the droplet are fixed at their crystallographic positions using a 200 kcal•mol -1 •Å -2 harmonic position restraint. Only those ionizable residues that fall within the mobile region (inner 85%) of the simulation sphere were ionized during the simulations, all other ionizable residues outside the mobile region were kept in their charge neutral states to avoid instabilities introduced by having charges located outside the explicit simulation sphere. Proton states of ionizable residues within the explicit simulation sphere, as well as histidine protonation patterns (both of which were validated by PROPKA 3. 1 and visual inspection), can be found in Table .