title: Supercooled liquid-like solvent in trypsin crystals: implications for crystal annealing and temperature-controlled X-ray radiation damage studies.
abstract: 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.
authors.affiliation: Institut de Biologie Structurale, Laboratoire de Biophysique Moléculaire, Grenoble, France. weik@ibs.fr
authors.forename: M
authors.initials: M
authors.lastname: Weik
authors.forename: A M M
authors.initials: AM
authors.lastname: Schreurs
authors.forename: H K S
authors.initials: HK
authors.lastname: Leiros
authors.forename: G
authors.initials: G
authors.lastname: Zaccai
authors.forename: R B G
authors.initials: RB
authors.lastname: Ravelli
authors.forename: P
authors.initials: P
authors.lastname: Gros
mesh_terms: D000818:Animals; D002417:Cattle; D015925:Cryopreservation; D003460:Crystallization; D003461:Crystallography / Q000379:methods*; D005615:Freezing; D011487:Protein Conformation / Q000528:radiation effects; D014357:Trypsin / Q000737:chemistry* / Q000528:radiation effects*
chemical_list: D014357:Trypsin