The complexes and free ligands were then solvated with TIP3P water, buffer molecules, and ions, to approximate the experimental conditions . To facilitate the calculation of relative binding enthalpies, the contents of each simulation box were kept identical, except for the choice of ligand, whether or not the protein was present, and the requirement for additional sodium ions to ensure electrical neutrality of the overall system (see below). Thus, each truncated octahedral simulation box, measuring 12 Å from the solute to the box edge for the complex and 23 Å for the free ligand, was populated with 6034 TIP3P waters, 6 HEPES molecules, and 17 NaCl, to approximate the 50 mM HEPES, 150 mM NaCl solution used in the ITC experiments . To match the pH 7.45 conditions of the experiments, the charges on the residues of Grb2 SH2, including the protonation states of the histidines, were determined with the H++ 3.0 server () . Based on the pKa values predicted by MarvinSketch 14.10.7.0, 2014, ChemAxon (), three different ionization states of HEPES were included (see Supporting Information). At pH 7.45, the total charges of the Grb2 SH2 protein, the 6 HEPES molecules, and each ligand are +3, -3, and -2, respectively. Thus, to neutralize the simulation systems, additional Na+ were added: 2 for the complex systems, and 5 for the ligandonly systems. In total, the simulated complex systems comprised approximately 20,050 atoms, while the ligand systems comprised approximately 18,400 atoms; the Grb2 SH2 domain has 1,653 atoms.