AN IVEM FOR HIGH RESOLUTION ELECTRONS CRYO-MICROSCOPY

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This proposal requests funding for a state-of-the-art Intermediate Voltage Electron Microscope (IVEM) with Field Emission Gun (FEG). We are specifically interested in purchasing an FEI Tecnai F20 with Gatan cold holder and 1K X 1K CCD imaging camera. This 200 kV instrument will complement our 9 year old CM12 and because of the high spatial and temporal coherence of the electron beam from the FEG, will provide significantly enhanced phase contrast transfer of image frequencies, between 12-4 angstrom resolution. Hence, the Tecnci F2O will become the centerpiece of the Biophysics Structural Electron Microscopy Facility and provide instrumental capabilities that will be needed for biological 3D EM during the first 10 years of the new millennium. The Tecnai F2O will allow us to pursue the following projects at significantly higher resolution: a) 3D studies of the ribosome-ER translocon with and without nascent chains, b) 3D studies of the Nuclear Pore Complex (NPC) in different transport-related configurations, c) combined 3D and molecular modeling studies of a histone-chaperone complex that mediates nucleosome assembly, d) 3D helical studies of pili which facilitate cellular attachment by pathogenic bacteria, e) tomographic studies of the yeast microtubule organizing center, the Spindle Pole Body (SPB), f) 3D structural studies of Low Density Lipoproteins (LDL), both alone and complexed with the LDL-receptor, g) structural studies of membrane receptor-ligand complexes (GM1-cholera toxin & insulin receptor- insulin), h) studies of isolated rafts of Apo B, lipid emulsions and lipid phases during bile formation and i) morphological analysis of distinct amyloid fibrils formed by immunoglobulin light chains and transthyretin. Overall, these projects will use structural EM to enhance our understanding of the assembly and function of macromolecular complexes and machines, that are localized at membranes and within nuclei. These nano-machines carry out fundamental roles in chromatin assembly, mitosis and the biogenesis and compartmental targeting of proteins and ribonucleic acid complexes. Furthermore, these studies will provide insights into cellular processes of lipid metabolism mediated by LDL and the formation of both normal and aberrant structures with medical relevance to bacterial colonization atherosclerosis and amyloidoses.