High resolution electron cryo-microscope at Brandeis University.

描述(由申请人提供)：我们申请资金购买一台200千伏的电子显微镜(EM)，配有场发射枪(FEG)和扫描单元(STEM)，一个液氮冷却样品架和一个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，包括最近聘用的一名初级教员。需求的增加还来自现有项目，这些项目需要收集大型数据集。新仪器将安装在我们现有的Brandeis EM设施中，并扩大其容量和能力。具体地说，STEM单元将使我们能够对淀粉样纤维进行每长度质量的测量，这是这些纤维的关键特征。大幅面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，包括对阿尔茨海默氏症A2肽形成的淀粉样纤维的3D结构分析、细胞质动力蛋白的结构和与其功率中风相关的构象变化、与调节器结合的肌动蛋白核仁形成蛋白和生长的肌动蛋白细丝的结构研究、病毒细胞进入，以及动粒的分子组织。所有这些项目都涉及与人类疾病或突出的人类病原体(病毒)的机制有关的严重疾病。A2纤维在阿尔茨海默病中的作用尚不清楚，纤维以及较小的聚集体可能导致在疾病过程中观察到的神经元死亡。这些纤维的许多形态的分子结构将由低温电子显微镜确定。这将阐明它们的作用，以及作为原纤维前体的较小聚集体的结构。胞质动力蛋白在细胞分裂、信号传递、细胞形态和极化细胞生长等方面发挥着重要作用。不同状态和微管结合的动力蛋白的结构将用电子冷冻断层扫描(Cryo-ET)来确定。许多关键的细胞功能也依赖于对肌动蛋白组装和拆解的精确控制，例如通过福尔马林。用单粒子低温电子显微镜测定了含有多种福尔明调节剂的福尔明斯的结构。此外，通过对附着在生长中的肌动蛋白细丝上的福尔马林进行冷冻，将研究福尔马林对肌动蛋白成核的精确机制。病毒进入细胞是病毒生命周期中的关键事件之一，不同的病毒采取不同的策略。细胞进入将使用一个基于通过表达黄病毒PrM和E蛋白分离出的“小病毒样颗粒”的模型系统来研究。这些都将通过单颗粒冷冻-EM与单颗粒、基于荧光的分析相结合来可视化。最后，动点的形成是有丝分裂细胞分裂和姐妹染色单体彼此忠实分离的中心。通过将X射线结晶学研究与较小组件的冷冻电子显微镜相结合，将更好地了解动点的3D结构(哈佛医学院正在进行一项使用电子断层扫描来显示完整动点的平行努力)。

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).