¶ Overview
The Highlight Mode is intended for comparing closely related structures where differences are subtle but biologically relevant. While changes in ligand conformation are usually easy to identify using standard visualization tools, small rearrangements in the protein—particularly in and around the binding site—are often difficult to detect visually.
Highlight Mode simplifies this analysis by allowing you to highlight, in a single click, all residues that differ from a reference structure. Differences can be based on sequence variations or on side‑chain movements exceeding a user‑defined RMSD threshold, making it immediately clear which regions have actually changed.
Typical use cases include:
- Identifying ligand‑induced local rearrangements of residues in the binding site.
- Comparing different constructs of the same protein to reveal construct‑dependent variations.
- Comparing homologous proteins across species to reveal binding‑site differences.
- Identifying point mutations and amino‑acid substitutions that alter binding‑site geometry or physicochemical properties.
Contents
¶ Highlight Mode Menu
The bulb icon on the side toolbar opens the Highlight Mode menu. There are 2 different modes:
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This mode focuses on the binding site and operates as described below only when the Workspace has a reference pocket. If no reference pocket is defined, the highlight behavior is identical to the Full Structure option.
The pocket mode is useful when studying binding site differences across targets and off-targets or comparing different conformations of the same target. It will display differences within the superposed pockets. Residues that differ between the structures are displayed using line and cartoon representations, while residues that are identical are hidden. The full protein chains remain visible as a thin putty representation to provide structural context. In addition, a surface for the reference binding site.
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¶ Full Structure
This mode highlights residue‑level differences across the entire structure. Residues that differ between the two structures are displayed using line and cartoon representations, while residues that are identical are hidden. The full protein chains remain visible as a thin putty representation to provide structural context.
Keep in mind that the structural overlay is optimized on the region used for the alignment, which can be either the pocket or a selected chain. As a result, Highlight Mode is most reliable for interpreting subtle differences within that aligned region. Outside of it, residues may appear as shifted simply because they are not part of the superposed area and therefore fall outside the scope of the comparison.
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¶ Other Options
The RMSD slider allows you to control the RMSD threshold used to compare matching residues. If the side-chain RMSD between 2 matching residues is higher than the selected threshold, that residue is treated as a difference.
The highlight mode can also be inverted to display only residues that are similar between the compared structures. In the current user interface, this option is available as a checkbox labeled “Show common part”. In the new user interface, it is accessed via the “Show Similar Regions” button.
¶ How Does 3decision Evaluate Residue Differences?
To determine which residues can be compared, Highlight Mode uses the first structure loaded in the 3D viewer as the reference. For each additional structure, the positions of the Cα (carbon‑alpha) atoms are compared against those of the reference structure. Residues whose Cα atoms fall within a defined spatial distance are considered equivalent and are compared to identify similarities or differences.
If no matching residue is found—for example due to missing residues, missing loops, or large conformational differences—this is treated as a difference between the two structures. Residues without a match are therefore highlighted as different.
Once residues are matched, they are compared in more detail. Residues will be highlighted as different not only when their sequence or side‑chain conformation differs beyond the chosen RMSD threshold, but also when atoms are missing in one structure, or when residues share the same residue name but contain a different set of atoms. This ensures that incomplete residues, construct differences, and atom‑level discrepancies are explicitly captured during comparison.
Note: As described above, Highlight Mode always uses the first structure loaded into the 3D viewer—that is, the first object listed in the state tree—as its reference. As a result, the Highlight Mode reference is not necessarily the same as the workspace reference.
In most cases, this does not cause issues, as Highlight Mode is typically used to compare two structures loaded directly into the viewer. However, if multiple structures are loaded and you wish to compare two structures that are not the Highlight Mode reference, you must adjust the structure order.
To do so, hide structures using the eye icon in the left collection panel until one of the structures you want to compare becomes the first entry in the state tree and is therefore used as the Highlight Mode reference.