HIGH PRESSURE CRYOCOOLING IN PREPARATION FOR 70S RIBOSOME CRYSTALS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The primary purpose of these experiments is to study aspects of the high pressure cryocooling process in preparation for cryocooling 70S ribosome crystals. This involves basic studies of the formation of high density amorphous(HDA) ice in crystals (Gruner group task) by application to model protein crystals, and data collection on pressure frozen 70S ribosome crystals provided by the Noller group. High pressure cryocooling will be applied in three different directions. In all cases, these experiments cannot be performed at rotating anode sources. 1. Mechanism study involving HDA ice. The mechanism of high pressure cryocooling was proposed involving high density amorphous (HDA) ice, which has 1.17 g/cm3 at 1 bar and 77 K. We would like to prove that HDA ice does form by high pressure cryocooling and affect crystal diffraction. We plan to collect solution/crystal diffraction images by increasing temperature gradually. 2. The structural changes induced by pressure are usually very small but sometimes not negligible. We recently observed that elastase showed a significant crystal unit cell change (a= 50 A --> a= 46.5 A) when pressure frozen and the structure was quite different than the ambient pressure one. More interestingly, the changed one is very similar to the structure induced by chemical perturbation. We plan to study elastase as a model system to understand pressure effects on proteins. 3. 70S ribosome crystals will be pressure frozen using various protocols and examined.