How would you validate a proposed ligand-binding pocket?

Why

Check whether the pocket exists in experimentally supported conformations and whether key residues are confidently modeled. Inspect side chains, protonation, metal ions, cofactors, waters and experimental ligand density if structures exist. Use comparisons across homologs and known ligands without assuming conservation proves binding.

Build a small mutagenesis panel designed to perturb pocket chemistry while preserving protein folding. Measure ligand binding directly with an appropriate biophysical method and separately test functional activity, thermal shift or target engagement. For example, a mutation that abolishes a fluorescence readout but also unfolds the protein cannot isolate a binding-site mechanism.

Include orthogonal assay formats, inactive analogs and rescue variants. Report a provisional pocket hypothesis until physical and mechanistic evidence converge.