A docking run can fail—or produce results that are difficult to interpret—because the protein structure was not ready for the workflow. Alternate atom positions, unwanted waters, missing atoms, and uncertain protonation are easy to overlook when a structure first opens. Cleaning up does not mean removing everything that is not part of the protein: it means deciding which issues matter for the task, then making and checking the appropriate changes.
This guide follows a practical path in SAMSON, from a quick cleanup to more targeted repairs. The key distinction is between preparing a structure by applying common cleanup steps and validating it by inspecting specific structural issues.
Start with the question your workflow needs answered
Before deleting anything, consider what the protein will be used for. A docking setup, a molecular dynamics system, and a structure-analysis task may not need exactly the same components. Waters, ions, cofactors, and ligands can be unnecessary for one workflow but relevant to another. SAMSON’s preparation tools let you remove these components, but the decision about what to keep should follow the scientific purpose of the model.
If the structure appears complete and you mainly need standard cleanup, use Home > Prepare. This one-step operation can remove alternate locations, delete selected ligands and other small molecules, strip waters, clear monatomic ions, and add hydrogens. It is a convenient starting point, not a substitute for checking whether the resulting model fits your particular workflow.
Understand what cleanup changes
Crystal structures may contain alternate locations: multiple recorded positions for an atom or group of atoms. The preparation step keeps the highest-occupancy atoms. When occupancy values are tied or absent, SAMSON prefers alternate location A when present, otherwise B, and so on. This resolves the alternatives into a single set of coordinates, which can make a structure easier to use downstream.
Hydrogens require a little more thought. Edit > Add hydrogens adds standard hydrogens at pH 7 for standard amino and nucleic acid residues, and uses valences for other atoms. If a specific protonation pH is important, the PDBFixer extension can add hydrogens at a specified pH. Also, adding hydrogens does not automatically mean the whole structure must always be minimized: the appropriate follow-up depends on the downstream method. For example, the documentation notes that AutoDock Vina needs only polar hydrogens for determining hydrogen bonds.
Validate instead of guessing
After basic cleanup, use Home > Validate to inspect issues that a one-step preparation may not resolve. The Structure Validation module can find alternate locations, flag bonds outside expected length ranges, identify non-standard residues, and highlight clashes and contacts. Reviewing the findings helps distinguish an actual problem from a feature you may need to preserve.
If side-chain clashes are present, the Rotamers editor can change side chains using a backbone-dependent rotamer library. Validation also includes utilities to merge nodes, reorder atoms in a connected component, and renumber atom serial numbers—useful when keeping atom and node lists organized for simulations.
When cleanup is not enough
A structure with missing residues or atoms needs a deeper repair than simply removing unwanted components. The PDBFixer extension can add missing residues based on SEQRES records, build missing heavy atoms and side chains, convert non-standard residues to standard equivalents, and resolve alternate locations. It can also remove water or heterogens, add hydrogens for a specified pH, build explicit-solvent water boxes, and build lipid membranes around membrane proteins.
For a single structure, these options provide a route to address specific gaps. For many files, Batch Protein Prepare can process structures in a folder, preserve the internal subfolder structure in the output, or download and prepare structures from PDB identifiers. It supports PDB, PDBx/mmCIF, MMTF, and MOL2 inputs for folder processing.
A compact pre-workflow checklist
- Decide which waters, ligands, cofactors, and ions are needed for your scientific question.
- Resolve alternate locations and inspect the result.
- Check for unusual bond lengths, non-standard residues, and steric clashes.
- Add hydrogens with the appropriate method and pH for your task.
- If residues or atoms are missing, use a repair workflow such as PDBFixer rather than assuming routine cleanup will rebuild them.
Preparation is most useful when it is deliberate: clean what is unnecessary, repair what is missing, and validate what could affect the next calculation. For the full workflow and tool details, read the SAMSON protein preparation and validation tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.