A protein structure can look perfectly reasonable in a molecular viewer and still cause trouble downstream. Alternate atom positions, unwanted waters or ions, missing hydrogens, and incomplete residues can complicate docking, simulation, or analysis. The challenge is knowing which cleanup is enough—and when the structure needs a deeper repair.
A useful approach is to treat protein preparation as a short triage: clean what is unnecessary, validate what is present, and rebuild only when something is actually missing. That keeps routine preparation efficient without confusing “cleaned” with “complete.”
Start with the question your workflow needs answered
Before changing the structure, consider what you are preparing it for. For a docking or simulation workflow, you may need to remove alternate locations, discard molecules that are not part of the intended system, and add hydrogens. If the protein has missing residues or atoms, a one-click cleanup is not the same as rebuilding them; that calls for a repair tool such as PDBFixer.
This distinction matters because preparation steps change the model. For example, removing a ligand or cofactor is appropriate only if it is not needed for the task. Likewise, water molecules may be stripped for a particular workflow, but their presence or absence should be a deliberate choice rather than an automatic assumption.
Use the quick pass for routine cleanup
In SAMSON, Home > Prepare combines several common cleanup actions. It can remove alternate locations, delete selected categories of small molecules, strip water, clear monatomic ions, and add hydrogens. This is a convenient starting point when the structure is broadly intact and you know which components your downstream task requires.
Alternate locations deserve a moment of attention. A structure may contain different modeled positions for the same atom. Preparation keeps the highest-occupancy position. If occupancies are tied—or absent—the selection follows the alternate-location label preference described in the tool: A when available, otherwise B, and so on. This provides a consistent choice, but it is still worth reviewing the structure when the alternative positions could affect the site you are studying.
Validate instead of guessing
When you want to inspect the structure before or after cleanup, open Home > Validate. The Structure Validation module can find alternate locations and bonds outside expected length ranges, identify non-standard residue aliases, and highlight clashes and contacts. These checks help turn a vague concern—“is this model ready?”—into specific issues you can review.
Validation is not the same as automatic repair for every problem. For instance, clashes between side chains may call for inspecting or changing side-chain conformations with the Rotamers editor. The module also includes tools to merge nodes, reorder atoms in a connected component, and renumber atom serial numbers—useful housekeeping when a workflow expects a tidy atom or node list.
To remove ligands, water, or ions manually, use Select > Biology and choose the relevant category, then erase the selection. To add standard hydrogens, use Edit > Add hydrogens. This adds hydrogens at pH 7 based on residue type for standard amino and nucleic acids, and based on valences for other structures. If you need hydrogens for a specific pH, use PDBFixer instead. Whether to minimize after adding hydrogens depends on the downstream task; it is not a universal requirement.
Escalate when atoms or residues are missing
If the protein has missing residues, missing heavy atoms, or incomplete side chains, use the PDBFixer extension rather than relying on routine cleanup. It can add missing residues based on SEQRES records, add heavy atoms and side chains, convert non-standard residues to standard equivalents, and add hydrogens at a specified pH. It can also resolve alternate locations, remove water or heterogens, and build explicit-solvent water boxes or lipid membranes.
PDBFixer can work with structures loaded in SAMSON and with PDB or mmCIF/PDBx files, including batch processing of files in a folder. For repeated work, Batch Protein Prepare can prepare structures in a folder or download and prepare structures from PDB identifiers.
A compact decision path
- Mostly complete structure? Use Home > Prepare for the cleanup steps your workflow actually needs.
- Unsure what needs attention? Run relevant checks in Home > Validate and review the findings.
- Missing residues or atoms, or need pH-specific hydrogens? Use PDBFixer for deeper repair.
- Preparing many structures? Consider Batch Protein Prepare, then review the resulting models as appropriate for your workflow.
The goal is not to apply every operation to every structure. It is to identify the specific problems that could affect the next step, make the necessary changes, and keep track of what was removed or rebuilt. For the complete walkthrough and tool details, see the SAMSON protein preparation and validation documentation.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.