A protein structure can look complete in a molecular viewer and still cause trouble downstream. Alternate atom positions, unwanted waters, missing hydrogens, or incomplete residues may complicate docking, simulations, and structure analysis. The challenge is not simply to “clean everything”; it is to identify which issues matter for your workflow and fix them without removing something important.
SAMSON brings common preparation and validation steps together, but a useful first habit is to distinguish routine cleanup from structural repair. Routine cleanup handles items such as alternate locations and hydrogens. Deeper repair is needed when atoms or residues are missing. Knowing which situation you have helps you choose the right tool rather than treating every structure the same way.
Start with a quick preparation pass
For a straightforward starting point, choose Home > Prepare. This applies several common operations in one step:
- Resolves alternate locations by retaining the highest-occupancy atoms. If occupancies are tied or absent, the selection follows the documented preference for alternate location A, then B, and so on.
- Removes ligands, including covalently attached ligands, cofactors, and other small molecules you do not need.
- Strips water molecules and clears monatomic ions.
- Adds hydrogens according to residue type for standard residues, or by valence for other structures.
That list also points to an important decision: “unneeded” depends on the question you are asking. A ligand may be irrelevant for one analysis but essential if you are studying a protein–ligand complex. Waters, cofactors, and ions can likewise be part of the system you intend to model. Review what the operation will remove and preserve anything required for your scientific goal.
Check before you move on
Preparation is not a substitute for inspection. Open Home > Validate to examine alternate locations, bond lengths, non-standard residues, and clashes. For alternate locations, use the Alt. locations tab, choose Find alternate locations, review the results, and then select Remove alt. locations if appropriate.
Validation can help expose problems that a quick cleanup does not solve. For example, the module can find bonds outside expected ranges and highlight steric clashes. If side-chain clashes need attention, the Rotamers editor can change side chains using a backbone-dependent rotamer library. The validation tools also include options for keeping atom and node lists organized, such as merging nodes, reordering atoms in a connected component, and renumbering atom serial numbers.
Hydrogens and protonation are not one-size-fits-all
Edit > Add hydrogens adds standard hydrogens at pH 7 for standard amino-acid and nucleic-acid residues, and uses valences for other structures. Select a substructure first if you want to limit where hydrogens are added. If your work requires hydrogens at a specific pH, use PDBFixer instead, which supports a specified pH.
Also consider what your next calculation actually requires. Some workflows may need minimization after hydrogens are added, but it is not always necessary. The documentation notes that AutoDock Vina, for example, needs only polar hydrogens for determining hydrogen bonds. Avoid adding extra steps automatically; choose them based on the downstream method.
When atoms or residues are missing
If the structure has missing residues or atoms, use the PDBFixer extension for a deeper repair. It can add missing residues based on SEQRES records, build missing heavy atoms and side chains, convert non-standard residues to standard equivalents, and add hydrogens for a specified pH. It can also remove water or heterogens, resolve alternate locations, and prepare explicit-solvent water boxes or lipid membranes. PDBFixer can work with structures loaded in SAMSON, PDB and mmCIF/PDBx files, and batches of files in a folder.
A practical sequence is therefore: decide what components your study needs, perform the appropriate cleanup, validate the result, and use a repair tool if the structure is incomplete. For many files, Batch Protein Prepare can apply the preparation steps to a folder or download structures from PDB identifiers.
Read the full SAMSON protein preparation and validation tutorial for detailed steps and related workflows.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.