A protein structure can look perfectly reasonable on screen and still contain details that complicate the next step: alternate atom positions, missing hydrogens, unexpected molecules, or incomplete residues. Discovering these issues only after a docking run or simulation has started can mean wasted time and confusing results. A short, deliberate preparation pass helps you see what is present, decide what needs attention, and avoid making changes your workflow does not require.
The key is to treat preparation as a sequence of decisions—not simply a command to delete everything except the protein. A ligand, ion, cofactor, or water molecule may be irrelevant to one task and important to another. First identify the purpose of the model; then clean, validate, and repair accordingly.
1. Start with the quick preparation pass
In SAMSON, Home > Prepare applies several common cleanup operations in one step. It can resolve alternate locations by retaining the highest-occupancy atoms, delete selected ligands and other small molecules, strip water, clear monatomic ions, and add hydrogens.
Before deleting molecules, consider what your next workflow needs. For example, if you are preparing a protein–ligand complex, deleting the ligand would remove part of the system you intend to study. Likewise, cofactors, ions, or waters may be relevant to a particular model. The practical takeaway: use the one-click operation for speed, but review its options in the context of the question you are asking.
2. Check alternate locations and geometry
Some structures contain multiple positions for atoms, often represented as alternate locations. Keeping all of these alternatives can leave downstream tools with an ambiguous or unexpected atom list. To inspect them, open Home > Validate, go to the Alt. locations tab, choose Find alternate locations, and review the results before selecting Remove alt. locations.
The default selection logic keeps the highest-occupancy atoms. When occupancies tie—or are absent—the documented behavior prefers alternate location A when available, then B, and so on. Knowing this rule makes the cleanup more transparent: it is not an arbitrary choice, but it is still worth checking the structure if a particular region matters to your analysis.
Structure Validation also helps identify bonds outside expected length ranges, non-standard residue names or aliases, and steric clashes or contacts. These checks do not automatically mean the model is unusable; they help you find places to inspect. If side-chain clashes need attention, the Rotamers editor can change side chains using a backbone-dependent rotamer library.
3. Add hydrogens with the workflow in mind
Edit > Add hydrogens adds standard hydrogens at pH 7, using amino- and nucleic-acid types for standard residues and valences for other atoms. Select a substructure first if you want to limit where the operation applies. If your modeling question requires a different pH, the PDBFixer extension can add hydrogens at a specified pH.
Adding hydrogens does not automatically mean the whole structure must be minimized. Whether minimization is needed depends on the downstream task. The documentation gives AutoDock Vina as an example where only polar hydrogens are needed for determining hydrogen bonds. Check the requirements of the method you plan to use rather than adding extra processing by default.
4. Escalate only when the structure needs repair
If residues or atoms are missing, routine cleanup may not be enough. 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 at a specified pH, and prepare explicit-solvent water boxes or lipid membranes.
For multiple structures, Batch Protein Prepare can process files in a folder or download structures by PDB identifier, then apply the same cleaning steps as Home > Prepare. Batch processing is useful for consistency, but it makes an initial review especially valuable: decide which components should be retained before applying the same operations across a collection.
A reliable preparation habit is simple: identify what your workflow needs, run the appropriate cleanup, inspect validation findings, and use deeper repair tools when residues or atoms are actually missing. Then continue to docking, simulation, or analysis with a clearer understanding of the model you are using.
Learn more in the SAMSON protein preparation and validation tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.