Acquisition of a Cryo-Electron Microscope

描述（由申请人提供）：要获得的特定冷冻电子显微镜（cryoEM）是JEOL jeem -2100，这是一台200千伏的透射电子显微镜（TEM），具有硼化镧（LaB6）发射器，完整的冷冻封装和用于断层扫描的台阶倾斜能力。这是最容易操作和维护的TEM之一，使其成为许多低温TEM设施的首选仪器。当设施扩大时，通常保留它作为训练工具，因为它在多用户环境中具有多功能性，并且修理和维护费用相对低廉。它比同类仪器更能满足一般研究环境的基本需要。结构研究提供了一种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，填补了核磁共振和x射线提供的原子分辨率与光学显微镜对细菌、整个细胞和细胞器等较大实体的可视化之间的空白。为了从低温电镜数据中获得三维结构，单粒子重建依赖于收集同一物体的不同二维视图，然后将其重建为三维地图。断层扫描聚焦于单个物体，通过倾斜舞台和拍摄来获得重建所需的不同视图

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.