AN IVEM FOR HIGH RESOLUTION ELECTRONS CRYO-MICROSCOPY

该提案要求为最先进的带场发射枪（FEG）的中压电子显微镜（IVEM）提供资金。我们特别有兴趣购买FEI Tecnai F20与Gatan冷保持器和1 K X 1 K CCD成像相机。这种200 kV仪器将补充我们9年的CM 12，并且由于FEG电子束的高空间和时间相干性，将提供显着增强的图像频率的相位对比转移，分辨率在12-4埃之间。因此，Tecnci F2 O将成为生物物理学结构电子显微镜设施的核心，并提供新千年头10年生物3D EM所需的仪器功能。Tecnai F2 O将使我们能够以更高的分辨率进行以下项目：a）具有和不具有新生链的核糖体-ER易位子的3D研究，B）不同转运相关构型的核孔复合物（NPC）的3D研究，c）介导核小体组装的组蛋白-伴侣蛋白复合物的组合的3D和分子建模研究，d）通过病原菌促进细胞附着的皮利的3D螺旋研究，e）酵母微管组织中心、纺锤体极体（SPB）的断层摄影研究，f）低密度脂蛋白（LDL）单独和与LDL受体复合的3D结构研究，g）膜受体-配体复合物（GM 1-霍乱毒素和胰岛素受体-胰岛素）的结构研究，h）胆汁形成期间分离的载脂蛋白B筏、脂肪乳剂和脂质相的研究，和i）由免疫球蛋白轻链和甲状腺素运载蛋白形成的不同淀粉样原纤维的形态学分析。总的来说，这些项目将使用结构EM来增强我们对位于膜和核内的大分子复合物和机器的组装和功能的理解。这些纳米机器在染色质组装、有丝分裂以及蛋白质和核糖核酸复合物的生物发生和隔室靶向中发挥重要作用。此外，这些研究将深入了解由LDL介导的脂质代谢的细胞过程以及与细菌定植动脉粥样硬化和淀粉样变性具有医学相关性的正常和异常结构的形成。

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.