Stage 1: Prepare 1. Obtaining the structure and the parameter file for the trypsin-benzamidine complex This section will prepare input files for the SEEKR2 simulation to determine the (un)binding kinetics between the receptor protein, trypsin, and the ligand molecule, benzamidine. The Amber molecular mechanics forcefield is used to parameterize the receptor-ligand complex. This tutorial assumes the user has some familiarity with AmberTools. If the user is new to AmberTools, we recommend visiting the Amber tutorials page () or reviewing the Amber manual () for a comprehensive understanding. Outline 3 shows the files we will require for the successful completion of the SEEKR2 tutorial on the trypsin-benzamidine complex.