When simulating ligand unbinding pathways in protein-ligand complexes, one surprisingly common source of difficulty isn’t always the algorithm or even the protein structure itself—it’s defining the sampling region. Many molecular modelers spend hours tuning energy parameters, refining force fields, and adjusting alignment, only to get unrealistic ligand escape routes or no paths at all. The shape and position of the sampling box used for active ligand atoms can dramatically affect the outcome of a simulation.
In the Ligand Path Finder app from SAMSON, the ligand’s conformational exploration is guided by a sampling box that defines where the system should attempt to move the active atoms of the ligand. This is especially important when using the ART-RRT method, which combines Rapidly-exploring Random Trees and ARAP (As-Rigid-As-Possible) modeling to explore unbinding paths.
What exactly is the sampling box?
The sampling box is a 3D Cartesian region in which selected active ARAP atoms of the ligand are allowed to move during the search for unbinding pathways. It constrains the search space and biases the path planner, helping it to find meaningful and plausible escape routes.
When defining the sampling box in the Ligand Path Finder app, you can customize the exact size and position of the box in each direction. A green box will appear around the ligand in the viewer to indicate this region visually.
Why is box size and orientation so important?
If the box is too small, there simply won’t be enough space for the ligand to find viable exit paths. If the box is misaligned with the most likely solvent-accessible tunnels or exit pathways, the algorithm might miss key regions, leading to unproductive searches or unrealistic conformational changes.
To avoid this, orient your protein-ligand system before defining the conformation. This ensures the sampling box—defined in Cartesian coordinates—covers the biologically relevant exit regions. For example, in the tutorial system involving Thiodigalactosid (TDG) bound to Lactose permease, the complex is aligned such that the sampling box biases the ligand motion toward the periplasmic side.
Tips for better sampling box setup:
- Visual inspection: Use the green box to verify that it encloses regions of biological interest, such as known tunnels or channels in the protein.
- Use alignment tools: Before setting the sampling box, use the Move or Align functionalities to align the system with principal protein-ligand axes.
- Don’t overconstrain: Allow sufficient volume for exploration, especially if the unbinding occurs via flexible or wide tunnels.
- Incrementally refine: Test with a larger box, observe the paths found, and refine progressively to exclude irrelevant directions.
When in doubt, check the docs
Getting the sampling box right can make or break a simulation. It’s not about being precise to the angstrom but rather about understanding the system’s geometry and ensuring enough room for the ligand to make meaningful exits. Explore the full documentation to learn about additional setup elements like active/fixed atoms, search parameters, and energy evaluations.
🔗 Read the full Ligand Path Finder tutorial here.
SAMSON and all SAMSON Extensions are free for non-commercial use. You can get SAMSON at https://www.samson-connect.net.