This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The primary focus of this initiative is on developing new approaches for preparing crystals to yield high-quality diffraction, thus enabling detailed structural analysis of challenging and extremely important questions of fundamental biological interest. Collaborative experiments will address two such biological problems in particular: (1) structural studies of a functional ribosome unit, in collaboration with Dr. Harry Noller (U.C. Santa Cruz) and (2) structural analysis of signaling proteins responsible for the programmed death of cells (apoptosis), in collaboration with Dr. Hao Wu (Cornell-Weill Medical College). In addition, we will investigate the use of pressure as a thermodynamic parameter to study the mechanisms of protein dynamics, function, and interaction, as well as seeking a fundamental understanding of how proteins assume and maintain their three dimensional structures;Dr. Sol Gruner will lead this effort. Three basic methodologies will be used: (1) Cryocooling crystals under high pressure for ambient pressure diffraction at cryogenic temperatures, (2) diffraction of pressurized crystals at physiological temperatures and high pressures using beryllium and diamond pressure vessels, and (3) application of novel flash-cooling and annealing techniques, using protocols developed by a Cornell technical collaborator, Dr. Rob Thorne. Cornell and MacCHESS have a long history of innovations in the field of crystal cryocooling, beginning with the development of cryoloops by T-Y Teng in 1985 [5]. Currently, groups led by Sol Gruner and Rob Thorne are actively investigating cryocooling techniques, with support from MacCHESS. Over the next five years, we, together with our collaborators, propose to focus on this area to develop apparatus and techniques for handling crystals under various conditions, particularly at high pressures. Our overall goals will be (1) to permit MacCHESS users to obtain the best possible diffraction from their crystals, (2) to increase the success rate for noble-gas phasing, and (3) to further investigate the dependence of protein structure on pressure and temperature. Dissemination of the new methodologies, and training of the larger MacCHESS user community, is occurring now, and will continue to occur concurrently with future developments. Pressure cryocooling apparatus development The pressure-cryocooling apparatus developed so far is a prototype that has worked well for initial experiments [18]. Briefly, crystals are picked up in a cryoloop with an attached magnetic steel wire in a droplet of Hampton NVH oil to prevent dehydration. These are loaded into the high pressure cryocooling apparatus consisting of commercial high pressure plumbing and pressure transducers. The apparatus containing the crystal is then pressurized with helium gas to pressures in the 100 - 200 MPa range. Once at high pressure, a magnetic constraint is released and the crystals fall down a length of high pressure tubing into a cold zone kept at LN2 temperature. The helium pressure is released and the crystals are thereafter handled at ambient pressure, in the same manner as normal flash-cryocooled crystals for cryocrystallographic data collection. The crystals are indefinitely stable as long as they are not allowed to warm above ~130? K. The process for Kr or Xe phasing is a bit more complex. First, crystals are loaded into the apparatus as in the previous paragraph. The crystals are then pressurized with Kr or Xe gas to 10 MPa. After an equilibration time, the compressed gas is released and the crystals are re-pressurized with helium. The apparatus is then pressurized to 100 ?200 MPa over the course of a few minutes, after which the magnetic constraint is released and crystals are dropped into a cold zone at liquid nitrogen temperature. With the publication of the pressure-cryocooling method [18], many crystallographers have expressed interest in trying it on their crystals, and as of December 2006 about two dozen groups have collaborated to do so. Results have been encouraging and are beginning to appear in the literature [12]. However, it has become clear that the existing apparatus needs to be improved for efficiency and safety and to extend experimental capabilities. Gas at several hundred MPa presents significant explosion hazards and must be handled very carefully. The prototype apparatus is housed in Dr. Gruner's lab in Cornell's Physics Department, and is not suitable for a general user facility. There have also been requests to reproduce the apparatus at the NSLS and the APS. Accordingly, as part of the dissemination mission of MacCHESS, we are including ease of reproduction as a goal in future redesigns of the apparatus. A redesigned cryocooling apparatus is now being commissioned (Fig. 30). Improvements include: (1) Pressure capability to 400 MPa. The old apparatus was limited to 200 MPa. (2) A more robust, larger, and easier to use safety enclosure to enable various cryocooling protocols. (3) Automation to reduce the time between Kr or Xe gas exposure and pressure cryocooling. (4) Modifications to be able to process more crystals at a time. The redesigned apparatus will be tested in Dr. Gruner's physics department lab, which will also be the site for experimentation to extend the technique and develop robust usage protocols (see below). We propose to build a second apparatus to be installed at CHESS for MacCHESS users using these protocols. The MacCHESS technical operations staff will be trained to operate this machinery. A third apparatus will likely soon be installed at the NSLS, and others may follow at other institutions. New protocols developed in Dr. Gruner's lab will be disseminated to other installations as part of the Mac- CHESS dissemination mission. High pressure gas involves significant safety issues, but we are confident that these can be dealt with in the CHESS user environment. Procedures have already been developed at CHESS for using a high pressure gas-loading apparatus for diamond anvil cells;the new apparatus will use some of the same gas-handling equipment and safety precautions. Protocols will need to be developed for inexperienced users. The pressure cryocooling apparatus at CHESS will be a research tool, used largely for crystallographically difficult proteins, i.e., those where other methods have not worked. Note that this effort is distinct from, yet synergistic with, an effort at CHESS (as a member of the High Throughput Center for Structural Biology, a Protein Structure Initiative center based at the Hauptmann-Woodward Institute in Buffalo, NY) to develop a high-throughput pressure cryocooling apparatus for crystal screening.
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