CRYO CORE

低温核心

• 批准号: 7507581
• 负责人: Peijun Zhang
• 金额: $ 50.72万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7507581
• 关键词: 3-Dimensional; Algorithms; Architecture; Back; Biological; Cell Communication; Cell Nucleus; Cells; Cellular Morphology; Cellular Structures; Complex; Condition; Confocal Microscopy; Crowding; Crystallography; Cytoplasm; Data; Discipline; Dose; Electron Microscope; Electron Microscopy; Electrons; Event; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Freezing; Frozen Sections; Genetic; HIV; HIV Infections; Image; Individual; Label; Life; Locales; Mammalian Cell; Methodology; Methods; Microscope; Microscopic; Molecular; Multiprotein Complexes; Nature; Nuclear Import; Organelles; Pathogenesis; Proteins; Radiation; Resolution; Roentgen Rays; Series; Signal Transduction; Specimen; Staging; Standards of Weights and Measures; Structure; System; Techniques; Technology; Temperature; Thick; Time; Tomogram; Viral; Viral Pathogenesis; Virion; Work; base; cell type; cellular imaging; computerized; cryogenics; data acquisition; density; design; electron tomography; fluorescence imaging; improved; insight; interest; intracellular protein transport; light microscopy; macromolecular assembly; macromolecule; novel; particle; pressure; protein localization location; single molecule; structural biology; time interval; tomography; tool

The emerging discipline of electron tomography provides new and unprecedented opportunities to determine three-dimensional cellular architecture at resolutions of ~50A or better, i.e., potentially high enough to identify individual macromolecules such as proteins in a 3-D volume of a cell289. Electron tomography is especially applicable for the structural analysis of cellular organelles and other macromolecular assemblies that are too heterogeneous to be investigated by NMR or X-ray crystallographic techniques or even electron microscope-based methods that involve averaging of multiple copies of the same object to improve signal-tonoise ratios. Electron tomography bridges the gap between high resolution structure determination of protein complexes by NMR or X-ray crystallographic techniques and single-particle living cell imaging by light microscopy using fluorescent probes. It extends the resolution of cellular imaging by one to two orders of magnitude over what is currently achieved using light microscopy. The fundamental principles underlying electron tomography and the strategy to use back-projection algorithms to extract 3-D information from a series of 2-D images recorded at different orientations were clearly articulated nearly four decades ago290'291. However, this field has seen a significant burst of activity in the last five years, principally propelled by the availability of tools for automated data acquisition using modern computerized microscopes. Efforts at imaging complex assemblies at room temperature as well as cryogenic temperatures have rapidly begun to provide many new insights into the 3-D architecture of cells289'292. During the immediate post-entry stages in HIV pathogenesis, the viral particle or, more precisely, its contents must traverse through the cytoplasm of the host cell prior to nuclear import of the pre-integration complex (PIC). Identification and characterization of these cellular events and viral and host interactions are central to understanding the molecular events that occur during HIV pathogenesis. NMR or X-ray crystallographic methods will provide high resolution structures of the viral and host protein complexes, while confocal microscopy in combination with fluorescent-labeling techniques allows tracking of viral particles and assessing the approximate localization of individual proteins in living cells. However, advancement of electron microscopic techniques, particularly electron tomography, will be essential to obtain higher resolution snapshots of the molecular arrangement of multiprotein complexes in the context of the host cellular structure and to provide the structural information necessary to fill the gap between NMR or X-ray crystallographic structural determination and live cell imaging. We, therefore, expect that our work to develop approaches for 3- D cellular imaging will have a direct impact on cellular and structural studies of HIV infection.

电子层析成像的新兴学科为新的和前所未有的机会提供了 在〜50a或更高的分辨率下确定三维蜂窝结构，即可能足够高 鉴定单个大分子，例如3-D体积的细胞289。电子断层扫描是 特别适用于细胞细胞器和其他大分子组件的结构分析 太异构了，无法通过NMR或X射线晶体学技术甚至电子进行研究 基于显微镜的方法，涉及平均相同对象的多个副本以改善信号量 比率。电子断层扫描桥接蛋白质的高分辨率结构测定之间的差距 通过NMR或X射线晶体学技术和单粒子活细胞成像的复合物 使用荧光探针显微镜。它将细胞成像的分辨率扩展到一到两个阶 使用光学显微镜当前实现的幅度。 电子断层扫描基础的基本原则和使用反向预测的策略 从不同方向记录的一系列2-D图像中提取3-D信息的算法显然是 阐明了将近四十年前的290'291。但是，该领域在最后一个活动中看到了大量活动 五年，主要是由于使用现代的自动数据获取工具所推动的 计算机显微镜。在室温和低温下对复杂组件进行成像的努力 温度已迅速开始为细胞的3-D结构提供许多新的见解。289'292。 在HIV发病机理的立即入学后阶段，病毒颗粒或更确切地说是 在核进口核进口之前，含量必须穿过宿主细胞的细胞质 复杂（图片）。这些细胞事件以及病毒和宿主相互作用的识别和表征是 了解在HIV发病机理期间发生的分子事件的中心。 NMR或X射线 晶体学方法将提供病毒和宿主蛋白复合物的高分辨率结构，而 共聚焦显微镜与荧光标记技术结合使用，可以跟踪病毒颗粒和 评估单个蛋白质在活细胞中的近似定位。但是，电子的发展 微观技术，尤其是电子断层扫描，对于获得更高的分辨率至关重要 在宿主细胞结构的背景下，多蛋白复合物分子布置的快照 并提供填补NMR或X射线晶体学之间空隙所需的结构信息 结构确定和活细胞成像。因此，我们期望我们的工作开发3-- D细胞成像将直接影响HIV感染的细胞和结构研究。