AN IVEM FOR HIGH RESOLUTION ELECTRONS CRYO-MICROSCOPY

这项提案要求为配备场发射枪(FEG)的最先进的中压电子显微镜(IVEM)提供资金。我们特别感兴趣的是购买FEI Tecnai F20，带有Gatan冷架和1K X 1K CCD成像相机。这台200千伏的仪器将补充我们已有9年历史的CM12，由于来自FEG的电子束具有高度的空间和时间一致性，因此将在12-4埃分辨率之间提供显著增强的图像频率的相位对比度转移。因此，Tecnci F2O将成为生物物理结构电子显微镜设备的核心，并提供新千年头10年生物3D EM所需的仪器能力。Tecnai f2o将使我们能够以更高的分辨率进行以下项目：a)带有和不带有新生链核糖体-ER转位的3D研究，b)不同运输相关构型的核孔复合体(NPC)的3D研究，c)介导核小体组装的组蛋白-伴侣复合体的3D和分子建模研究，d)促进病原菌细胞附着的毛状物的3D螺旋研究，e)酵母微管组织中心、纺锤体极体(SPB)的断层研究，f)低密度脂蛋白(LDL)的3D结构研究，既有单独的，也有与低密度脂蛋白受体复合的，(G)膜受体-配体复合体(GM1-霍乱毒素和胰岛素受体-胰岛素)的结构研究，h)分离的载脂蛋白B筏的研究，脂肪乳剂和胆汁形成过程中的脂类阶段的研究，以及i)由免疫球蛋白轻链和转甲状腺素形成的不同的淀粉样纤维的形态分析。总体而言，这些项目将使用结构EM来加强我们对位于膜和核内的大分子复合体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体的生物发生和隔室靶向方面发挥着基础作用。此外，这些研究将深入了解低密度脂蛋白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相关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.