This page explains how Highlight Mode decides which residues to show. It works in two steps: it matches residues between each structure and the reference, then it compares each matched pair.
Highlight Mode always compares against the Workspace reference structure, so the reference must be loaded and visible in the 3D viewer.
For each residue, it looks at the position of the Cα atom. Residues of the two structures whose Cα atoms are within 2 Å of each other are matching residues, and only those are compared in detail.
Matching is purely spatial, so it depends on the superposition. If two structures are not superposed, almost nothing matches and almost everything is flagged as different.
A residue is shown as different when any of these is true:
Complete flips of symmetric groups are not counted as differences, for example the ring of Phe and Tyr, or the carboxylate of Asp and Glu.
Show Similar Regions is the opposite of Show Differences: it hides everything that differs from the reference. Only matching residues of the same amino acid type, with an RMSD below the threshold, are shown.
The threshold decides how much movement counts as a change. A low value flags small side-chain shifts, which can be noise at lower resolution. A high value only flags large rearrangements. The default is 2 Å; set it with the RMSD slider.
In the legacy UI, Pocket mode only compared residues present in both pockets, and flagged residues present in only one pocket as different. This limitation is fixed in the new UI.