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abstracts
Almost all diffraction experiments in structural biology are done at cryogenic temperature to mitigate radiation damage with cryoprotected crystals to avoid solvent crystallization and its harmful consequences: formation of ice rings, loss of diffraction and exaggeration of mosaicities. Crystallographers often protect crystals with glycerol rather than with genuine conditions whose search is time consuming. Moreover, cryo-protection has detrimental consequences: Soaking somewhat destabilizes crystals, mosaicity expend upon cooling, cryo-agents may interact with macromolecules and in extreme cases, cryo-conditions are destructive. High pressure (HP) cooling is an alternative method which consists of flash-cooling cryoprotectant-free samples under 200 MPa of Helium [1]. The solvent is directly turned into high density amorphous (HDA) ice avoiding water crystallization and preserving/improving the sample quality owing to the absence of cryo-agents and to HAD-ice properties. We have developed a HP-cooling system and its associated methodology paying attention to user-friendliness and throughput aspects through a new pluggable Spine compatible base-pin and to its handling-toolkit [2]. The machine allows vitrifying solution with 5% glycerol for cooling diluted bio-objects, but importantly, it is very effective for cryoprotectant-free crystals since the sole mother liquor components act as anti-freeze agents. Its capability was demonstrated with test crystals and resulting structures appear isomorphous to those deposited in the PDB since the structural changes are limited to flexible loops. Nevertheless, pressure is a key thermodynamic variable which produces structural modifications associated with different conformational protein substates or reaction coordinates [3], and we provide examples of HP structures which address biological questions. On the basis of the HP-cooling technique, we have designed a novel of pressure cell for gases of biological interest. The method takes advantage of thermodynamic properties which allow liquefying pressurized gases; crystals are soaked in the gas phase prior to be flash-frozen in the liquid phase without pressure relaxation. This technique has been applied to noble gas derivatives and oxygen sensitive macromolecules.
abstracts
Cryo-cooling of protein crystals is not often used in neutron crystallography. However cryo-temperatures are used to block the reaction processes at specific intermediate stages, and this has been widely used in X-ray studies (1); (2). In order to develop this area for joint neutron/X-ray applications, trypsin was chosen as a suitable system for which its interaction with the substrate succinyl-Ala-Ala-Pro-Arg-p-nitro-aniline could be studied (3). Here the neutron developments were carried out in parallel with complementary X-ray techniques, and also using in crystallo UV-visible and Raman spectroscopy. Various strategies for doing this have been tested. The installation of an N2-gas-cryostream system on the D19 single crystal diffractometer at the Institut Laue Langevin (ILL) and the development of a new carboloop mounting system, has opened new avenues to perform cryo-cooling experiments using a neutron source. Preliminary data collection carried out at the ILL and at the European Synchrotron Radiation Facility (ESRF), have confirmed the feasibility of the approach. A full description of the experimental procedures and results will be presented. As part of this a new carboloop mounting system has been developed that is suitable for both X-ray and neutron data collection. These mounts resolve the problems of activation and hydrogen incoherent scattering in neutron experiments We describe the use of these and their advantages over conventional X-ray mounting systems - including compatibility with standard magnetic goniometer heads and resistance to cryogenic temperatures.
