Two protein structures can look broadly similar and still disagree where it matters: around a binding site, a flexible loop, or a conserved helix. A whole-protein superposition is useful, but it can be a poor answer to a local question. When many residues contribute to the fit, the overall alignment may not place a particular region on top of its counterpart.
A practical way to avoid that mismatch is to separate two questions: Which residues correspond? and How well do the structures match in the region I care about? SAMSON’s Protein Aligner lets you inspect sequence correspondence, select aligned residues, and superimpose structures using either the whole protein or a selected region.
Start with sequence correspondence
Load the structures you want to compare, then open Home > Align. The tutorial demonstrates the workflow with hemoglobins 1DLW and 1RTX, fetched through Home > Fetch. Before comparing, consider whether the structures contain water, ligands, or alternate locations that should be removed for your analysis; SAMSON’s Protein Preparation & Validation guide covers cleanup.
In Protein Aligner, you can align sequences by structural model or by chain. The chain-based option is useful when working with oligomers, where a model contains multiple chains. Sequence alignment gives you a way to inspect corresponding residues before moving the structures. You can also highlight residue properties, such as similarity and polarity, to help spot conserved positions or differences.
Hovering over a residue shows its name and ID and highlights it in the 3D viewport. Clicking a residue selects it and its aligned partners. This interaction is useful when you want to connect a sequence-level observation to the corresponding positions in the structures.
Use the global fit as a first check
To superimpose the complete proteins, make sure no residues are selected, then click Align to this on the row for the model you want to use as the reference. The other model’s button reports the RMSD for the resulting fit. In the example, it can show a value such as 3.27 Å.
That number describes the result of the alignment you performed; it does not, by itself, tell you whether a particular helix or binding-site region is well matched. Treat the global fit as a useful overview, then inspect the structural feature that motivated the comparison.
Fit the region that answers your question
If a local region is the focus, select corresponding residues in both sequences and use the alignment button next to that selection. The structures are then superimposed based on the selected residues rather than the entire protein. In the tutorial, the first 20 residues are selected because the corresponding alpha-helices appear similar but are not well aligned after the whole-protein fit.
This is not a replacement for the global comparison; it is a different comparison with a different purpose. A local fit helps answer questions such as “Do these helices adopt a similar arrangement?” A global fit helps answer “How do these structures compare overall?” Keeping the selected region in mind also makes RMSD easier to interpret: the value belongs to the fit you asked the software to perform.
A compact checklist
- Prepare the structures if extra solvent, ligands, or alternate locations would interfere with your comparison.
- Choose sequence alignment by model or by chain, depending on the organization of your proteins.
- Inspect residue correspondence and select the region relevant to your question.
- Run a whole-protein fit for an overview, then a selected-residue fit for a local comparison.
- Interpret the reported RMSD in the context of which residues were used for the alignment.
For visual clarity, ribbon models can be added through Visualization > Visual model > Ribbons. Creating separate ribbon models for the proteins lets you color them differently, making an overlay easier to inspect.
For the complete workflow and additional examples, see the Protein Aligner tutorial.
Note: SAMSON and all SAMSON Extensions are free for non-commercial use. Get SAMSON.