A docking setup can look ready and still contain details that trip up the next step: alternate atom positions, unwanted waters or ions, missing hydrogens, or incomplete residues. The difficult part is not simply cleaning a structure; it is knowing which problems need a quick cleanup and which need a more substantial repair.
A useful approach is to treat preparation as a short decision process. Start with the intended workflow, make only the changes it calls for, and validate the result before moving on. This helps avoid spending time repairing issues that do not matter for your task—or deleting molecules that you meant to keep.
First, decide what kind of preparation you need
If the protein looks complete and you mainly need routine cleanup, SAMSON’s Home > Prepare command brings several common operations together. It can remove alternate locations, delete selected categories of small molecules, strip waters, clear monatomic ions, and add hydrogens. This is a convenient starting point, but the options matter: ligands, cofactors, waters, and ions can be relevant to some models. Review what your downstream workflow requires before removing them.
One detail about alternate locations is worth understanding. A structure may contain alternative positions for atoms, often represented by alternate-location labels. The preparation tool keeps the highest-occupancy atoms; when occupancies are tied or absent, it uses the available labels in a defined order, preferring A when present. This turns multiple possible positions into one selected set for the prepared structure.
Use validation to inspect, not just clean
Cleaning operations and validation answer different questions. Preparation applies selected changes; Home > Validate helps you inspect specific structural issues. For alternate locations, open the corresponding tab, find the locations, review the list, and then remove them if appropriate. Validation can also identify bonds outside expected length ranges, non-standard residue aliases, and clashes or contacts.
That review step is useful because a warning is not automatically a reason to edit the model. For example, if a clash is between side chains, the documentation points to the Rotamers editor as a way to change side-chain conformations using a backbone-dependent rotamer library. The key is to inspect the issue and choose a targeted correction rather than treating every check as a command to remove or alter atoms.
Hydrogens: choose the method for the job
Edit > Add hydrogens adds standard hydrogens at pH 7, using residue types for standard amino and nucleic acids and valences for other atoms. You can select a substructure first to limit where hydrogens are added. If your setup needs hydrogens for a particular pH, the PDBFixer extension provides a pH-specific option.
Do not assume that adding hydrogens always means you must minimize the structure afterward. The right follow-up depends on the workflow. The documentation gives AutoDock Vina as an example where only polar hydrogens are needed for determining hydrogen bonds. Check the requirements of your own downstream tool before adding extra preparation steps.
When a quick cleanup is not enough
If residues or atoms are missing, routine cleanup cannot supply all the missing structure. The PDBFixer extension can add missing residues based on SEQRES records, add missing heavy atoms and side chains, convert non-standard residues to standard equivalents, and add hydrogens at a specified pH. It can also process files in batches, as well as structures loaded in SAMSON. For suitable systems, it offers options to build explicit-solvent water boxes or lipid membranes.
For many files, Batch Protein Prepare can apply the routine cleaning steps to a folder of structures or download structures from supplied PDB identifiers. It supports PDB, PDBx/mmCIF, MMTF, and MOL2 inputs for folder preparation, and preserves the internal subfolder structure in the output.
A compact pre-workflow checklist
- Identify which waters, ions, ligands, and cofactors your task needs before removing them.
- Check for alternate locations and decide whether to keep the highest-occupancy position.
- Inspect bond lengths, residue labels, and clashes when relevant.
- Add hydrogens using the method and pH appropriate to your workflow.
- If residues or atoms are missing, use a repair workflow rather than relying on routine cleanup alone.
Preparation is not a single universal recipe: it is a way to make the structure consistent with the work you plan to do next. For the full procedures and tool details, see the SAMSON protein preparation and validation tutorial.
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