Increasingly, the frontiers of cell biology focus on elucidating the control of cellular shape and organization, the nature and function of cellular organelles, and the role of complex protein machines. Such project areas also define a new frontier for structural biology where the goals are to elucidate not the structure of individual molecules, but structures of molecular and supramolecular complexes as well as organelles. EM methods such as single particle reconstruction and EM Tomography (EMT) are ideally suited to the analysis of such large structures. EMT alone is appropriate for large unique structures such as chromosomes, centrosomes, the cytoskeleton, neuronal organization, etc. EMT alone is appropriate for large unique structures such as chromosomes, centrosomes, the cytoskeleton, neuronal organization, etc. While great strides in the development of EMT have been made at UCSF, our instrumentation is inadequate to meet the demands of current problems in structural cell biology. Our goals are to acquire a new microscope (Phillips 300kV FEG He stage) equipped with an energy filter and a 2Kx2K CCD camera. As a first step in this expensive process we request the purchase of a Gatan Imaging Energy Filter (GIF) and a Gatan 2K x2K pixel CCD camera. This equipment will be immediately useful on our existing microscope and readily transferable to the new microscope. Acquisition of the energy filter and 2Kx2K CCD will create a facility for the collection of high resolution EM Tomography data that is absolutely unique in the US and perhaps the world. This new equipment will allow us to do 1) higher resolution tomography on thick biological samples, 2) image larger samples, 3) collect high resolution image data for single particle reconstruction using the CCD and 4) collect high quality diffraction data from 2D crystals. These benefits result from the use of the GIF to remove inelastic and multiple inelastic scattered electrons and having a larger format CCD that is significantly better suited for imaging at 200-300keV. The equipment will be attached to our pre-existing Philips 300keV electron microscope in the Electron Microscope Laboratory of the Department of Biochemistry at UC San Francisco. This facility is available to the entire campus as well as to outside users. All of the core users except two are currently funded by the NIH, and all have extensive experience in electron microscope. The core users plus the supervisor of the EM lab will form an advisory committee to oversee use of the instrument and to set policy. Use by other regional and national users will be encouraged in the time remaining after core investigator use. UC San Francisco will provide space and staff support (including software) for operating the equipment.
