I want to calculate the binding free energy of ligand in the protein-ligand complex. I have two ligands in the complex could you please guide me how can I apply restraints on both the ligands with protein?
If I understand correctly, you already have your molecular dynamics results and you want to carry out binding free energy studies using MMPBSA method. So if your protein-ligand complex is present in your system and remains stable during the equilibrium phase you do not have to establish restrictions on ligands to calculate the energy, that would have to be established before performing your simulation if it were the case of forcing a position for a protein, ligand or group of atoms.