To determine the anchor points for milestoning simulations in the SEEKR2 framework, we choose the appropriate CV for the trypsin-benzamidine complex. Here, the distance between the center of mass (COM) of the binding site of the trypsin protein and the ligand serves the CV, followed by determining the anchor points (Figure ). Subsequently, we establish concentric spherical milestones around the binding site, using midpoints between successive anchor points as their radii. Through SMD simulations, the benzamidine ligand is slowly extracted from the binding site, capturing trajectory snapshots at each anchor point. Once the ligand is pulled out entirely and we have the saved structure files, a SEEKR2 file tree is created where SEEKR2 simulations will occur.