Protein–protein docking can feel like searching for a key in a room full of possible orientations. A broad search explores many ways a ligand can approach a receptor, but it can also take longer and return many poses that are difficult to interpret. If you have a reasonable idea of the binding region or starting orientation, restricting the search can make the calculation more focused—while keeping in mind that overly strict constraints may exclude the pose you are looking for.
This workflow uses the Hex Extension in SAMSON. It is especially useful when you want to explore docking orientations for two prepared protein structures and have some information that can guide the search.
Start with structures you can trust
Before docking, make sure the receptor and ligand structures are suitable for the calculation. Remove atoms with alternate locations, waters, monatomic ions, and ligands if they are not part of the system you intend to dock. Add hydrogens when needed. If your structure already has hydrogens added according to a chosen protonation state, do not modify them casually: changing protonation can change the model you are evaluating.
Missing residues or heavy atoms, such as side-chain atoms, may need attention before docking. The SAMSON documentation points to the PDBFixer Extension for fixing proteins and adding hydrogens for a specified pH. Once the structures are ready, assign one protein as the receptor and the other as the ligand in Hex. Saving the ligand’s initial conformation before running the search is also useful if you later want to compare runs with different parameters.
Use what you know to narrow the search
Hex can search over protein orientations using spherical coordinates. With the Range angles sampling method, you can limit the rotational range of the receptor and ligand around the intermolecular axis connecting the proteins. A range of 180 degrees leaves the full sphere accessible; a smaller range focuses the search around the starting orientation.
If you know approximately where the binding site is, first move and orient the ligand near that site. Then, in Advanced parameters, set the receptor and ligand range angles to values that reflect your confidence. The tutorial demonstrates 45 degrees for each. Treat this as an example, not a universal setting: a narrow range is only helpful if the starting placement is informative. If the orientation is uncertain, a wider search may be more appropriate.
You can also constrain the ligand’s twist around the intermolecular axis. These controls reduce the number of orientations considered, which can reduce search time and help limit irrelevant predictions. They do not establish that a pose is biologically correct; they define which possibilities the calculation is allowed to explore.
Choose parameters with the trade-offs in mind
The correlation type determines what the docking calculation evaluates, such as shape alone or shape combined with electrostatics. Electrostatics may be useful when the proteins have complementary formal charges, but the documentation advises against using electrostatics when docking DNA or RNA molecules.
Hex can use a fast, lower-resolution Steric scan before a more detailed Final search. The first stage quickly discards many orientations; the second examines the retained candidates more finely. The tutorial notes that a Steric scan order of 16–18 is sufficient in many cases, and describes combinations such as 16 for the scan and 25 for the final search as practical starting points. Higher final-search orders can be more time-consuming. The suggested order also depends on the structure: 30 is recommended for high-resolution crystal structures expected to change little on binding, while 25 may be more suitable for model-built or more flexible structures.
Post-processing can help sort candidates. A bumps counter reports steric clashes between non-bonded heavy atoms, and the results table can be used to filter solutions by clash count. Molecular-mechanics refinement is described as an alpha-stage option in Hex; its rigid-body refinement may help when conformational changes are expected to be small, but should not be used when large conformational changes are expected.
Review poses instead of relying on one score
After the run, inspect the clustered modes in the Results tab. You can view the best-scoring mode per cluster or all modes, select a row to restore its ligand conformation, and create a conformation in the document for further examination. Compare candidates visually and, where appropriate, examine contacts, hydrogen bonds, and surrounding residues. SAMSON’s Protein-Ligand Interaction Analyzer and Hydrogen Bond Finder are examples of tools that can support this follow-up analysis.
The practical lesson is simple: prepare the structures carefully, use prior knowledge to set sensible search limits, and treat docking results as candidates for analysis—not as a final answer. For the full workflow and parameter details, see the Protein docking with Hex tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.