A docking setup can look ready and still contain issues that affect the workflow: alternate atom positions, unwanted waters or small molecules, missing hydrogens, or geometry problems. Finding these before docking is easier than diagnosing unexpected results later. The key is to prepare deliberately: clean what is unnecessary, retain components your study depends on, and validate the structure before moving on.
This workflow focuses on protein preparation in SAMSON. It is useful when you have loaded a structure and want to check it before docking, simulation, or other modeling tasks.
Start with a quick preparation
For a structure that appears reasonably complete, use Home > Prepare. This single-step tool can remove alternate locations, delete selected ligands, strip water, clear monatomic ions, and add hydrogens.
These operations are convenient, but they are not all automatic decisions about what your scientific question requires. For example, a ligand, cofactor, ion, or water molecule may be relevant to the system you intend to study. Review what should remain before deleting anything. Preparation is not simply about making a structure smaller; it is about making it appropriate for the next step.
Alternate locations are another common source of uncertainty. A structure may contain multiple modeled positions for an atom. The preparation tool keeps the highest-occupancy position. If occupancies are equal or absent, it prefers location A when available, then B, and so on. This gives you a consistent choice, but it is still useful to inspect unusual cases when the local structure matters to your analysis.
Validate rather than guess
For a more controlled review, open Home > Validate. In the alternate-locations tab, choose Find alternate locations to inspect the results, then use Remove alt. locations when you are ready to resolve them. Validation can also help identify bonds outside expected length ranges, non-standard residue names, and clashes or contacts.
A clash is a warning to investigate, not necessarily a cue to edit blindly. If side-chain clashes need correction, the Rotamers editor can change side chains using a backbone-dependent rotamer library. The validation tools also include utilities for organizing atom and node lists, such as merging nodes, reordering atoms in a connected component, and renumbering atom serial numbers.
Add hydrogens with the workflow in mind
Use Edit > Add hydrogens to add standard hydrogens at pH 7. For standard amino and nucleic acid residues, placement is based on residue type; for other structures, it is based on valence. Select a sub-structure first if you want to limit where hydrogens are added.
Hydrogen placement and protonation are related, but they are not always the same preparation question. If you need hydrogens for a particular pH, the PDBFixer extension supports specifying pH. Also consider what the next method requires: the documentation notes that AutoDock Vina needs only polar hydrogens for determining hydrogen bonds, so minimizing the entire system after adding hydrogens is not always necessary. Follow the requirements of your downstream workflow rather than treating minimization as an automatic step.
When a quick cleanup is not enough
If residues or atoms are missing, use the PDBFixer extension for a deeper repair. It can add missing residues based on SEQRES records, add missing heavy atoms and side chains, convert non-standard residues to standard equivalents, resolve alternate locations, and add hydrogens at a specified pH. It also offers options for building explicit-solvent water boxes or lipid membranes around membrane proteins.
For repeated work, Batch Protein Prepare can apply the same cleaning steps to files in a folder or download structures from supplied PDB identifiers. It supports PDB, PDBx/mmCIF, MMTF, and MOL2 inputs for folder preparation, and can preserve the folder’s internal subfolder structure in the output.
A useful stopping point
Before moving on, ask three questions: Have I kept the molecules needed for my study? Have I resolved or reviewed alternate locations and structural issues? Does the hydrogen and repair strategy match the requirements of my next workflow? A short check now can prevent avoidable confusion later.
For the full tutorial and details on each tool, see the SAMSON protein preparation and validation documentation.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.