Cryo-Electron Microscopy and Image Analysis of Plant Viruses

植物病毒的冷冻电子显微镜和图像分析

• 批准号: 9206305
• 负责人: Timothy Baker
• 金额: $ 33.5万
• 依托单位: Purdue Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9206305&HistoricalAwards=false
• 关键词: Cryo; Electron; Microscopy; Image; Analysis

This study involves the coordinated application of cryo-electron microscopy (cryoEM), three-dimensional image reconstruction, and X-ray diffraction methods to address fundamental questions about the overall morphology, composition and function of plant viruses. These studies will focus on structural analyses of three plant viruses: cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus (CCMV), and barley yellow dwarf virus (BYDV). Complexes of CPMV with two separate monoclonal Fab fragments will be studied to investigate the detailed interactions between viruses and antibodies as well as the organization of the single-stranded RNA (ssRNA) in CCMV (and also brome mosaic virus). Work with BYDV aims to localize the factor(s) responsible for aphid transmissibility. In addition, studies will be made to coordinate cryoEM and X-ray data to learn how to correct for the effects of the microscope contrast transfer function. This should allow for reconstructed density maps that give a more accurate definition of protein and nucleic acid distributions and also provide the potential to attain higher resolution. %%% These studies provide a powerful way to examine protein-protein and protein-nucleic acid interactions which are involved in establishing virus stability and dictating viral assembly and disassembly (uncoating). The cryoEM techniques are ideally suited for studying large macromolecules and complexes that have proved difficult or "impossible" to crystallize and examine by diffraction methods. Long term goals are to examine and understand virus-antibody interactions, examine protein and nucleic acid organization, and develop enhanced methodology for studying virus structures with complementary biophysical techniques. The plant viruses are excellent model systems for these studies because i) they are readily and inexpensively obtained in large, highly-purified quantities, ii) they are not human pathogens, and iii) their molecular biology and genetics are well characterized.

本研究涉及低温电子的协同应用 显微镜（cryoEM），三维图像重建，以及 X射线衍射方法来解决以下基本问题 植物整体形态、组成和功能 病毒 这些研究将侧重于结构分析， 三种植物病毒：豇豆花叶病毒（CPMV），豇豆 褪绿斑驳病毒（CCMV）和大麦黄矮病毒 （BYDV）。 CPMV与两个单独的单克隆Fab的复合物 碎片将被研究，以调查详细的 病毒和抗体之间的相互作用以及 CCMV中单链RNA（ssRNA）的组织（以及 雀麦花叶病毒）。 与BYDV合作旨在将 影响蚜虫传播性的因素。 此外，本发明还提供了一种方法， 将进行研究，以协调冷冻EM和X射线数据， 如何校正显微镜对比度的影响 传递函数 这应该可以重建密度 更精确地定义蛋白质和核酸的图谱 酸分布，也提供了实现 更高的分辨率。 %%% 这些研究提供了一种强有力的方法来检查蛋白质-蛋白质 和蛋白质-核酸相互作用， 建立病毒稳定性并指示病毒组装， 拆卸（无涂层）。 冷冻电镜技术是理想的 适用于研究大的高分子和复合物， 被证明是困难的或“不可能”的结晶和检查， 衍射方法 长期目标是检查和 了解病毒-抗体相互作用，检查蛋白质， 核酸组织，并开发增强的方法， 研究病毒结构与互补的生物物理 技术. 植物病毒是很好的模型系统， 这些研究是因为i）它们容易且便宜 大量、高纯度地获得，ii）它们不是 人类病原体，以及iii）它们的分子生物学和遗传学 很好地描述了。