Water Networks Repopulate Protein–Ligand Interfaces with Temperature

High‐resolution crystal structures highlight the importance of water networks in protein–ligand interactions. However, as these are typically determined at cryogenic temperature, resulting insights may be structurally precise but not biologically accurate. By collecting 10 matched room‐temperature and cryogenic datasets of the biomedical target Hsp90α, we identified changes in water networks that impact protein conformations at the ligand binding interface. Water repositioning with temperature repopulates protein ensembles and ligand interactions. We introduce Flipper conformational barcodes to identify temperature‐sensitive regions in electron density maps. This revealed that temperature‐responsive states coincide with ligand‐responsive regions and capture unique binding signatures that disappear upon cryo‐cooling. Our results have implications for discovering Hsp90 selective ligands, and, more generally, for the utility of hidden protein and water conformations in drug discovery.