Instrumentation for upgrading cryoEM and single particle analysis capabilities

用于升级冷冻电镜和单颗粒分析能力的仪器

• 批准号: 7594874
• 负责人: JACK D GRIFFITH
• 金额: $ 31.01万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7594874
• 关键词: 3-Dimensional; Area; Arts; Binding; Cells; Characteristics; Collection; Complex; Comprehensive Cancer Center; Computers; Core Facility; Cryoelectron Microscopy; DNA Repair; DNA biosynthesis; Engineering; Equipment; Eye; Filament; Freezing; Funding; HIV; HIV vaccine; Herpesviridae; Housing; Ice; Image; Individual; Malignant Neoplasms; Membrane; Methods; Microscope; Negative Staining; Neurodegenerative Disorders; North Carolina; Nucleic Acids; Oncogenic; Operative Surgical Procedures; Patients; Preparation; Process; Proteins; Repair Complex; Research; Research Personnel; Research Project Grants; Resolution; Robotics; Roentgen Rays; Sampling; Structure; System; Training; Translating; Transmission Electron Microscopy; United States National Institutes of Health; Universities; Ursidae Family; Vaccines; Viral Proteins; Virus; Work; abstracting; base; coronin protein; image processing; instrument; instrumentation; macromolecular assembly; medical schools; novel strategies; particle; protein structure; public health relevance; skills; three dimensional structure; voltage

DESCRIPTION (provided by applicant): Instrumentation for upgrading cryoEM and single particle analysis capabilities Abstract Examination of the 3 dimensional structure of large macromolecular assemblies often of megadalton mass or greater remains beyond the reach of X ray or NMR methods. Transmission electron microscopy however provides a means of directly visualizing large protein and nucleic acid-protein assemblies. Newer approaches in which images of hundreds to thousands of individual structures are digitized and processed using computer based averaging routines provide a means of generating 3-D structures with resolution in the range of 2.0 nm when samples are imaged by negative staining. Further structural detail can often be obtained by imaging the samples frozen in ice using cryoEM methods and this can increase the resolution into the range of 1.0 nm. The basic skill for carrying out these methods are in place at the University of North Carolina Medical School and both 3-D averaging of negative stained images and cryoEM preparation of samples has been carried out. To advance the studies of a group of active NIH funded investigators, several critical pieces of equipment are needed so that state of the art work in this area can be carried out. A key item is a Vitrobot computer controlled robotic freezing instrument which will make it possible to freeze sets of grids all with the same characteristics for transport to a higher voltage FEG equipped microscope until such an instrument is available in the Research Triangle area. In addition to this instrument, a new cryotransfer system is requested as well as two new cameras of the 120 kV TEM which are needed for collection of large numbers of images for processing. The research projects which will utilize this instrumentation include studies of DNA replication complexes (Griffith), DNA repair complexes (Ramsden and Sancar), protein and DNA-protein filaments formed by Herpes virus proteins (Griffith, Damania, Raab-Traub), Coronin 1B protein filaments in the cell (Bear), membrane bound oncogenic proteins (Raab-Traub), protein filaments related to neurodegenerative diseases (Dokholyan), protein structures in the eye (Costell), and new engineered viruses being developed as vaccines against HIV (Johnston). The instrumentation will be housed in the Lineberger Comprehensive Cancer Center EM core facility and its administration, upkeep, and training in its use will be the responsibility of this facility which has been in operation for 27 years. Public Health Relevance: Instrumentation for upgrading cryoEM and single particle analysis capabilities Narrative The requested equipment will expand research efforts for multiple investigators at the University of North Carolina at Chapel Hill. These investigators are working to understand the cellular mechanisms involved in Cancer, HIV vaccines and Ophthalmologic and Neurodegenerative diseases. This understanding can then be translated into patient treatment.

描述（由申请人提供）：用于升级低温电子显微镜和单粒子分析能力的仪器摘要：检查通常具有兆道尔顿质量或更大质量的大型大分子组件的三维结构，这是X射线或核磁共振方法无法达到的。然而，透射电子显微镜提供了一种直接观察大蛋白和核酸-蛋白组合的方法。更新的方法是将数百到数千个单个结构的图像数字化，并使用基于计算机的平均程序进行处理，当样品通过负染色成像时，提供了一种生成分辨率在2.0 nm范围内的3-D结构的方法。进一步的结构细节通常可以通过使用冷冻电镜方法对冰冻样品进行成像来获得，这可以将分辨率提高到1.0 nm的范围内。北卡罗莱纳大学医学院已经掌握了实施这些方法的基本技能，并且已经对阴性染色图像进行了三维平均处理，并对样品进行了冷冻电镜制备。为了推进一组活跃的NIH资助的研究人员的研究，需要几个关键的设备，以便在这个领域进行最先进的工作。一个关键项目是Vitrobot计算机控制的机器人冷冻仪器，它将使冻结具有相同特征的网格集成为可能，以便运输到一个更高电压的FEG配备的显微镜，直到这样的仪器在研究三角地区可用。除了该仪器外，还需要一套新的冷冻转移系统和两台新的120千伏透射电镜摄像机，用于收集大量图像进行处理。利用该仪器的研究项目包括DNA复制复合体（Griffith）、DNA修复复合体（Ramsden和Sancar）、疱疹病毒蛋白（Griffith, Damania, Raab-Traub）、细胞中的冠状蛋白1B蛋白细丝（Bear）、膜结合致癌蛋白（Raab-Traub）、与神经退行性疾病相关的蛋白细丝（Dokholyan）、眼睛中的蛋白质结构（Costell）、以及正在开发的新的工程病毒作为艾滋病毒疫苗（Johnston）。该仪器将被安置在Lineberger综合癌症中心EM核心设施中，其管理，维护和使用培训将由该设施负责，该设施已经运行了27年。所要求的设备将扩大北卡罗来纳大学教堂山分校多名研究人员的研究工作。这些研究人员正在努力了解癌症、艾滋病毒疫苗、眼科和神经退行性疾病的细胞机制。这种理解可以转化为对病人的治疗。