Acquisition of a Cryo-Electron Microscope

获得冷冻电子显微镜

• 批准号: 8246870
• 负责人: Susan Hafenstein
• 金额: $ 60万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8246870
• 关键词: 3-Dimensional; Bacteria; Binding Sites; Cells; Complement; Complex; Cryoelectron Microscopy; DNA; DNA-Directed RNA Polymerase; Data; Data Collection; Diabetic Nephropathy; Electron Microscope; Environment; Family Picornaviridae; Grant; Image; Imagery; Internet; Lanthanum; Maps; Melanosomes; Organelles; RNA replication; Replication Initiation; Research; Resolution; Roentgen Rays; Rous sarcoma virus; Series; Staging; Structure; Techniques; Training; Virus; Virus Receptors; Zebrafish; computerized data processing; instrument; light microscopy; mutant; particle; podocyte; polypeptide; protein complex; receptor; reconstruction; repaired; success; three dimensional structure; tomography; tool; transmission process; two-dimensional; viral RNA

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.

描述（由申请人提供）：要获取的特定冷冻电子显微镜（冷冻）是JEOL JEM-2100，它是200 kV的传输电子显微镜（TEM），具有灯笼硼化物（LAB6）发射极，完整的冷冻套件和舞台倾斜能力。该TEM是最容易操作和维护的TEM之一，使其成为许多冷冻TEM设施的选择，作为首款获得的工具。当设施扩展时，通常将其保留为培训工具，因为它在多用户环境中具有多功能性，并且相对便宜地维修和维护。它的功能胜于相当的工具，可以满足一般研究环境的基本需求。一项结构性研究提供了一种有力的直接可视化工具，可以指导和补充其他研究方法。冷冻物可以在亚细胞水平上进行三维（3D）成像，从而填补了NMR和XRARE提供的原子分辨率之间的间隙，以及通过细菌，全细胞和细胞器等较大实体的光学显微镜可视化。为了从冷冻数据中获得3-D结构，单个粒子重建依赖于收集同一对象的不同二维视图，然后将其重建为3-D映射。层析成像侧重于一个对象，并通过倾斜舞台并采取行动获得重建所需的不同观点 一系列2-D图像。 PI和COPI的可视化受试者广泛地获得了此收购赠款范围。包括多蛋白质复合物和两个不同的DNA蛋白质复合物。小多肽结合位点将在RNA聚合酶上可视化。将检查Rous肉瘤病毒的组装中间体，[Picornavirus]复制期间形成的膜网和病毒RNA复制起始复合物。将研究病毒受体复合物，包括同时与受体和受体相互作用的病毒的结构。 [使用断层扫描，将量化黑色素体的数量，并在突变体和野生型斑马鱼中进行完善。最后，糖尿病性肾病中的足细胞微环境和结构将通过层析成像可视化。我们确定9个实验室，并描述12个项目，准备使用冷冻单粒子重建和层析成像技术来收集数据和数据处理。