© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Interrogating fragment libraries by X-ray crystallography is a powerful strategy for discovering allosteric ligands for protein targets. Cryocooling of crystals should theoretically increase the fraction of occupied binding sites and decrease radiation damage. However, it might also perturb protein conformations that can be accessed at room temperature. Using data from crystals measured consecutively at room temperature and at cryogenic temperature, we found that transient binding sites could be abolished at the cryogenic temperatures employed by standard approaches. Changing the temperature at which the crystallographic data was collected could provide a deliberate perturbation to the equilibrium of protein conformations and help to visualize hidden sites with great potential to allosterically modulate protein function. Too cool to show up: The common practice of cryocooling crystals was found to mask transient protein conformations. Data collected on a single crystal revealed ligand binding to a cryptic site at room temperature that was hidden at cryogenic temperature. These results contradict thermodynamic expectations and provide a method for discovering allosteric sites with great potential for modulating protein (mal)-function.