CRYO CORE

低温核心

基本信息

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
项目类别：
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

项目摘要

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.

电子断层扫描这一新兴学科提供了前所未有的新机遇以约 50A 或更高的分辨率（即可能足够高）确定三维细胞结构识别单个大分子，例如细胞 3D 体积中的蛋白质289。电子断层扫描是特别适用于细胞器和其他大分子组装体的结构分析它们太异质，无法通过 NMR 或 X 射线晶体学技术甚至电子技术进行研究基于显微镜的方法，涉及对同一物体的多个副本进行平均以改善信噪比比率。电子断层扫描弥补了蛋白质高分辨率结构测定之间的差距通过 NMR 或 X 射线晶体学技术分析复合物，并通过光进行单颗粒活细胞成像使用荧光探针的显微镜检查。它将细胞成像的分辨率提高了一到两个数量级其规模超过了目前使用光学显微镜所达到的水平。电子断层扫描的基本原理和使用反投影的策略从不同方向记录的一系列 2D 图像中提取 3D 信息的算法很清楚近四十年前提出的290'291。然而，该领域在过去的几年里出现了显着的活动爆发五年来，主要是由使用现代自动化数据采集工具的可用性推动的电脑显微镜。在室温和低温下对复杂组件进行成像的努力温度已经迅速开始为细胞的 3D 结构提供许多新的见解289'292。在 HIV 发病机制的进入后阶段，病毒颗粒，或者更准确地说，它的在预整合进入核之前，内容物必须穿过宿主细胞的细胞质复杂（PIC）。这些细胞事件以及病毒和宿主相互作用的鉴定和表征是对于理解 HIV 发病机制期间发生的分子事件至关重要。 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，同时共聚焦显微镜与荧光标记技术相结合，可以追踪病毒颗粒和评估活细胞中单个蛋白质的大致定位。然而，电子技术的进步显微技术，特别是电子断层扫描，对于获得更高分辨率至关重要宿主细胞结构背景下多蛋白复合物分子排列的快照并提供必要的结构信息，以填补 NMR 或 X 射线晶体学之间的空白结构测定和活细胞成像。因此，我们期望我们的工作能够开发 3- D 细胞成像将对 HIV 感染的细胞和结构研究产生直接影响。