Acquisition of a Cryo-Electron Microscope

描述（由申请人提供）：拟采购的特定冷冻电子显微镜（cryoEM）是一台JEOL JEM-2100，这是一台200 kV透射电子显微镜（TEM），配有硼化镧（LaB 6）发射器、完整的冷冻包和用于断层扫描的载物台倾斜能力。这种TEM是最容易操作和维护的仪器之一，使其成为许多低温TEM设备的首选仪器。在设施扩大时，它经常被保留作为培训工具，因为它在多用户环境中具有多功能性，而且维修和维护费用相对较低。它的功能比同类仪器更好，以满足一般研究环境的基本需求。结构研究提供了一个强大的直接可视化工具，可以指导和补充其他研究方法。CryoEM允许在亚细胞水平上进行三维（3D）成像，填补了NMR和X射线提供的原子分辨率之间的空白，并通过光学显微镜对细菌，全细胞和细胞器等较大实体进行可视化。为了从cryoEM数据获得3D结构，单粒子重建依赖于收集相同对象的不同二维视图，然后将其重建为3D图。断层扫描聚焦于单个物体，通过倾斜载物台并取 一系列二维图像PI和CoPI对这一收购赠款的可视化主题范围很广。包括多蛋白质复合物和两种不同的DNA-蛋白质复合物。小的多肽结合位点将在RNA聚合酶上显现。劳斯肉瘤病毒的组装中间体，[小核糖核酸病毒]复制过程中形成的膜网，以及病毒RNA复制起始复合物将被检查。将研究病毒-受体复合物，包括同时与受体和辅助受体相互作用的病毒的结构。[使用断层扫描，将量化黑素体的数量，并在突变体和野生型斑马鱼中细化3D结构。最后，糖尿病肾病中的足细胞微环境和结构将通过断层扫描可视化。这些项目的多样性，每一个选择，因为成功的可能性展示了冷冻电子显微镜技术的强大应用。我们确定了9个实验室，并描述了12个项目准备使用cryoEM单粒子重建和断层扫描技术的数据收集和数据处理。

DESCRIPTION (provided by applicant): The specific Cryo-Electron Microscope (cryoEM) to be acquired is a JEOL JEM-2100 that is a 200 kV Transmission Electron Microscope (TEM), with a Lanthanum Boride (LaB6) emitter, a full Cryo package and a stage-tilting capability for tomography. This TEM is one of the easiest to operate and maintain making it the choice of many cryo TEM facilities as a first instrument to acquire. It is often retained as a training instrument when facilities expand because it has a reputation for versatility in a multi-user environment and is relatively inexpensive to repair and maintain. It functions better than comparable instruments to fulfill the basic needs of a general research environment. A structural study offers a powerful tool of direct visualization that can guide and complement other research approaches. CryoEM allows three-dimensional (3D) imaging at the subcellular level, filling a gap between the atomic resolution provided by NMR and Xray, and visualization by light microscopy of larger entities such as bacteria, whole cells and organelles. To obtain 3-D structures from cryoEM data, single particle reconstructions rely on collecting different two dimensional views of the same object that are then reconstructed into a 3-D map. Tomography focuses on a single object and obtains the different views necessary for reconstruction by tilting the stage and taking a series of 2-D images. The subjects for visualization by the PI and CoPI's of this acquisition grant range broadly. Included are multi-protein complexes and two different DNA-protein complexes. The small polypeptide binding sites will be visualized on an RNA polymerase. The assembly intermediates of Rous sarcoma virus, the membranous webs formed during [picornavirus] replication, and a viral RNA replication initiation complex will be examined. Virus-receptor complexes will be studied, including the structure of a virus interacting with a receptor and a co-receptor simultaneously. [Using tomography the number of melanosomes will be quantified and the 3D structure refined in mutant and wild type zebrafish. Finally, the podocyte microenvironment and structure in diabetic nephropathy will be visualized by tomography.] The diversity of these projects, each chosen because of the likelihood of success showcases the powerful application of cryo-electron microscopy techniques. We identify 9 labs and describe 12 projects ready for data collection and data processing using cryoEM single particle reconstruction and tomography techniques.