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)：将获得的特殊低温电子显微镜(CryoEM)是JEOL JEM-2100，这是一种200千伏的传输电子显微镜(TEM)，具有硼化镧(LaB6)发射器、完整的低温封装和层析成像的工作台倾斜能力。这台瞬变电磁仪是最容易操作和维护的设备之一，使其成为许多低温瞬变电磁法设备的首选设备。当设施扩大时，它经常被保留作为一种培训工具，因为它在多用户环境中具有多功能性的名声，而且维修和维护相对便宜。在满足一般研究环境的基本需求方面，它的功能比同类仪器更好。结构研究提供了一个直接可视化的强大工具，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较大实体(如细菌、整个细胞和细胞器)的可视化之间的空白。为了从CryoEM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，然后将其重建成三维地图。断层扫描聚焦于单个对象，通过倾斜舞台和拍摄获得重建所需的不同视图 一系列2-D图像。这项收购授权的PI和COPI的可视化对象范围很广。其中包括多蛋白质复合体和两种不同的DNA-蛋白质复合体。小的多肽结合位点将在RNA聚合酶上可视化。将检查Rous肉瘤病毒的组装中间体、在[微小核糖核酸病毒]复制过程中形成的膜网以及病毒RNA复制起始复合体。将研究病毒受体复合体，包括同时与受体和辅助受体相互作用的病毒的结构。[利用断层扫描技术，突变斑马鱼和野生斑马鱼的黑素小体数量将被量化，3D结构将得到优化。最后，断层扫描将显示糖尿病肾病的足细胞微环境和结构。]这些项目的多样性，每个项目都是因为成功的可能性而选择的，展示了低温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个准备好使用低温EM单粒子重建和断层扫描技术进行数据收集和数据处理的项目。