Supercooled liquid-like solvent in trypsin crystals: implications for crystal annealing and temperature-controlled X-ray radiation damage studies

The study of temperature‐dependent physical changes in flash‐cooled macromolecular crystals is pertinent to cryocrystallography and related issues such as crystal annealing, X‐ray radiation damage and kinetic crystallography. In this context, the unit‐cell volume of flash‐cooled trigonal and orthorhombic trypsin crystals has been monitored upon warming from 100 to 200 K and subsequent re‐cooling to 100 K. Crystals of both forms were obtained under the same crystallization conditions, yet they differ in solvent content and channel size. An abrupt non‐reversible unit‐cell volume decrease is observed at 185 K in orthorhombic and at 195 K in trigonal crystals as the temperature is increased; this result is consistent with ultra‐viscous solvent leaving the crystals. Concomitant appearance of ice rings in the diffraction patterns suggests that the transported solvent forms crystalline ice. These results demonstrate that solvent in flash‐cooled protein crystals is liquid‐like near its crystallization temperature, as has been proposed, yet controversially discussed, for the case of pure water. The use of mineral oil prevents the unit‐cell volume decrease in trigonal but not in orthorhombic crystals. The observation of liquid‐like solvent has implications in the development of annealing protocols and points a way to the rational design of temperature‐controlled crystallographic studies that aim either at studying specific radiation damage or at trapping enzymatic intermediate states.