When modeling ligand unbinding pathways in protein systems, small oversights can lead to big blind spots — literally. One of the most frequent and easy-to-make mistakes is misdefining the sampling region, also known as the sampling box. Fortunately, SAMSON’s Ligand Path Finder App provides an interactive way to understand and fine-tune this key feature.
Let’s say you’ve spent time carefully preparing your protein-ligand structure, set up the Universal Force Field, and defined your ligand atoms. Everything looks good. But then, when you run the planner, the ligand doesn’t exit the binding site in a way that looks physically plausible, or worse: it doesn’t find an exit at all. Before you assume it’s a problem with the algorithm, ask yourself: did you check the sampling box?
What is the sampling box?
The sampling box is the 3D region within which the Ligand Path Finder samples motions of the ARAP active atoms of the ligand. In simple terms, this box guides the possible directions in which the ligand can move. Too small or misaligned, and likely exit paths can be cut off completely. Too large, and you risk wasting compute effort in irrelevant areas of conformational space.
Visualizing and customizing the sampling region
In SAMSON, defining the sampling box is a visual and customizable step. Here’s how it works:
- After setting ligand and ARAP atoms, head to the Set the sampling region box.
- The app gives you a suggested box that encloses both ligand and protein atoms.
- You’ll see a bright green box in the Viewport. This is your sampling region.
- Manually adjust the size and position to bias ligand motion toward a biologically relevant direction.
For instance, in the case of lactose permease (LacY), the tutorial aligns the system along the Z-axis so that a sampling box extension along Z biases motion toward the periplasmic side — the biologically accurate exit path. Here’s what that looks like:
Best practices
- Align the system carefully: Use SAMSON’s move and alignment tools to ensure your protein-ligand complex is oriented with respect to the sampling dimensions.
- Use biological knowledge: If you know where the ligand exits (e.g., periplasmic vs. cytoplasmic side), bias your sampling region accordingly.
- Preview early: Use small test runs to see whether sampling leads to expected unbinding behavior before committing to longer simulations.
Why it matters
The sampling box is not just a parameter — it’s a form of guidance. It can prevent misleading results and dramatically reduce computation time by focusing the search. Even minor tweaks can impact which paths are found, and therefore, which hypotheses you can test computationally.
This often-overlooked step is particularly useful in workflows involving challenging binding pockets, such as deep or cryptic pockets in GPCRs or kinases, where straightforward escape routes may be rare.
To learn more about sampling box setup and the full unbinding pathway workflow, visit the original Ligand Path Finder documentation page.
SAMSON and all SAMSON Extensions are free for non-commercial use. You can download SAMSON at https://www.samson-connect.net.