CRYOELECTRON MICROSCOPE AND CRYO-ULTRAMICROTOME

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Funds are requested for the purchase of a high resolution cryo-electron microscope. Research proposals of the users require the ability to observe particulate and sectioned material in the frozen-hydrated state. The microscope will also be available for low dose and conventional microscopy. Users with peer-reviewed NIH funding have been investigating structures at the molecular level using negative staining and shadowing techniques. These approaches have now been largely superseded by observation of unstained specimens in the frozen-hydrated state, which preserves them in an essentially native form. This approach is necessary in order to answer questions without staining, fixation or drying artifacts and because is capable of providing significantly improved resolution in many cases. It is also a powerful technique for observing time resolved structural changes that can be captured by combining rapid freezing with, for example, the release of metabolites from caged precursors. Projects requiring the use of this technique include studies of the molecular structure of muscle filaments, hemoglobin crystals, DNA-protein interactions and coated vesicles. The cryo-electron microscope will be the first at the University of Massachusetts Medical Center. The acquisition of this instrument is essential to provide for the current needs of users and for the initiation of new directions in projects that are now impossible to realize. This technology will provide a natural and powerful complement to the structural techniques that are already in use on this campus, including X-ray crystallography, 3D light microscopic studies of cellular structure, high resolution NMR and conventional and freeze-substitution and freeze fracture electron microscopic studies of subcellular structure.