High resolution electron cryo-microscope at Brandeis University.

布兰迪斯大学高分辨率电子冷冻显微镜。

• 批准号: 7822272
• 负责人: NIKOLAUS GRIGORIEFF
• 金额: $ 221.38万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-07 至 2010-09-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7822272
• 关键词: Actins; Address; Alzheimer' s Disease; Amyloid Fibrils; Arts; Binding; Biological Assay; Biological Models; Cell Shape; Cell division; Cell physiology; Cells; Cessation of life; Characteristics; Collection; Complex; Data; Data Set; Disease; Dynein ATPase; E protein; Electron Microscope; Electronics; Electrons; Event; Faculty; Film; Flavivirus; Fluorescence; Funding; Guns; Housing; Human; Kinetochores; Lead; Length; Life Cycle Stages; Light; Liquid substance; Measurement; Microfilaments; Microscope; Microtubules; Mitotic; Molecular; Molecular Structure; Morphology; Neurons; Nitrogen; Play; Power stroke; Research; Resolution; Roentgen Rays; Role; Scanning; Sister Chromatid; Specimen; Structure; Universities; Viral; Virus; Virus-like particle; base; cell growth; detector; electron tomography; human disease; imaging detector; instrument; medical schools; member; particle; pathogen; peptide structure; polarized cell; stem; three dimensional structure; tomography

DESCRIPTION (provided by applicant): We are requesting funds for a 200 kV electron microscope (EM) with a field-emission gun (FEG) and scanning unit (STEM), a liquid-nitrogen-cooled specimen holder and a state-of-the-art large-format electronic image detector (CMOS camera). The primary reason for the need of this new instrument is the increased demand on our current instruments from new projects and users, including a recently hired junior faculty member. The increased need also comes from existing projects that require collection of large data sets. The new instrument will be housed in our existing Brandeis EM facility and expand its capacity and capabilities. Specifically, the STEM unit will enable us to perform mass-per-length measurements on amyloid fibrils, a key characteristic of these fibrils. The large-format detector offers an alternative to recording data on film, thus accelerating research. Projects benefiting from the new instrument are broad in scope and include the 3D structural analysis of amyloid fibrils formed by Alzheimer's A2 peptide, the structure of cytoplasmic dynein and conformational changes associated with its power stroke, structural studies of the actin nucleator formin bound to regulators and growing actin filaments, viral cell entry, and the molecular organization of kinetochores. All of these projects address serious disorders associated with human disease, or the mechanism of prominent human pathogens (viruses). The role of A2 fibrils in Alzheimer's disease is not well understood and fibrils, as well as smaller aggregate may lead to the neuronal death observed in the course of the disease. The molecular structure of a number of morphologies of these fibrils will be determined by cryo-EM. This will shed light on their role, as well as the structure of the smaller aggregates, which act as fibril precursors. Cytoplasmic dynein plays a major role in cell division, signaling, cell shape, and polarized cell growth. The structure of dyneins in different states and bound to microtubules will be determined using electron cryo-tomography (cryo-ET). Many critical cellular functions also depend on the precise control of actin assembly and disassembly, for example by formins. The structure of formins in complex with several formin regulators will be determined by single particle cryo-EM. Furthermore, the precise mechanism of actin nucleation by formins will be investigated by cryo-ET of formins attached to growing actin filaments. Viral cell entry is one of the key events in the viral life cycle, and different viruses employ different strategies. Cell entry will be studied using a model system based on "small virus-like particles" isolated by expression of the flavivirus prM and E proteins. These will be visualized by single particle cryo-EM, combined with a single-particle, fluorescence-based assay. Finally, the formation of kinetochores is central to mitotic cell division and the faithful separation of the sister chromatids from each other. A better understanding of the 3D structure of kinetochores will be achieved by combining X-ray crystallographic studies with cryo-EM of smaller subassemblies (a parallel effort to visualize intact kinetochores using electron tomography is underway at Harvard Medical School).

描述（由申请人提供）：我们正在申请资金用于200千伏电子显微镜（EM），带有场发射枪（FEG）和扫描单元（STEM），液氮冷却样品架和最先进的大幅面电子图像探测器（CMOS相机）。需要这种新仪器的主要原因是新项目和用户对我们现有仪器的需求增加，包括最近聘请的初级教员。增加的需求还来自需要收集大型数据集的现有项目。新仪器将被安置在我们现有的Brandeis EM设施中，并扩大其容量和能力。具体来说，STEM装置将使我们能够对淀粉样蛋白原纤维进行质量/长度测量，这是这些原纤维的一个关键特征。大画幅探测器提供了在胶片上记录数据的另一种选择，从而加速了研究。受益于新仪器的项目范围广泛，包括由阿尔茨海默氏A2肽形成的淀粉样原纤维的3D结构分析，细胞质动力蛋白的结构及其与动力行程相关的构象变化，与调节因子和生长的肌动蛋白丝结合的肌动蛋白核蛋白formin的结构研究，病毒细胞进入和着丝点的分子组织。所有这些项目都涉及与人类疾病有关的严重失调，或主要人类病原体（病毒）的机制。A2原纤维在阿尔茨海默病中的作用尚不清楚，原纤维以及较小的聚集体可能导致疾病过程中观察到的神经元死亡。这些原纤维的许多形态的分子结构将由低温电镜测定。这将阐明它们的作用，以及充当原纤维前体的较小聚集体的结构。细胞质动力蛋白在细胞分裂、信号传导、细胞形态和细胞极化生长中起重要作用。利用电子冷冻断层扫描（cryo-ET）测定不同状态和与微管结合的动力蛋白的结构。许多关键的细胞功能也依赖于对肌动蛋白组装和拆卸的精确控制，例如形成蛋白。用单粒子低温电子显微镜分析了双胍调节剂配合物的结构。此外，我们还将利用附着在生长中的肌动蛋白细丝上的形成蛋白的冷冻et来研究形成蛋白形成肌动蛋白的确切机制。病毒进入细胞是病毒生命周期的关键事件之一，不同的病毒采用不同的策略。细胞进入将使用基于“小病毒样颗粒”的模型系统进行研究，该模型系统是通过表达黄病毒prM和E蛋白分离出来的。这些将通过单颗粒低温电镜观察，结合单颗粒荧光分析。最后，着丝点的形成是有丝分裂细胞分裂和姐妹染色单体相互忠实分离的核心。通过结合x射线晶体学研究和较小亚组件的冷冻电镜，可以更好地了解着丝点的三维结构（哈佛医学院正在进行利用电子断层扫描观察完整着丝点的平行努力）。