(ii) Parameterization of the receptor-ligand complex In the SEEKR2 framework, to estimate the binding and unbinding of receptor-ligand bimolecular complexes, one of the molecules is the ligand that needs to be parameterized separately. One method to accomplish this is to use the Antechamber program in AmberTools. Antechamber requires a structure file specific to the small molecule to perform the parameterization. In our case, we will utilize a PDB file that contains only the benzamidine structure, which has been parametrized using a semi-empirical method and the generalized AMBER force field. Although semi-empirical methods for assigning charges are quick and convenient, they may not provide the most accurate results for assigning partial charges to a molecule. It is advisable to explore more precise levels of quantum calculations to obtain partial charges, such as Hartree Fock with Density Function Theory (HF-DFT) or Møller-Plesset 2 (MP2) calculations within a charge model such as RESP or CHELPG. However, these calculations involving higher levels of quantum theory require dedicated quantum calculation software like Gaussian, GAMESS, ORCA, etc. Incorporating parameters from quantum calculation software is a complex topic that is beyond the scope of this tutorial. To save a separate PDB structure of the ligand molecule (benzamidine.pdb) from the given receptor-ligand complex (trypsin_benzamidine_init.pdb), we execute the following command in the terminal.