X-ray Determination of Proteins and Viruses

蛋白质和病毒的 X 射线测定

• 批准号: 9986266
• 负责人: Michael Rossmann
• 金额: $ 79.5万
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9986266&HistoricalAwards=false
• 关键词: ray; Determination; Proteins; Viruses

9986266Rossmann, M.X-ray diffraction and cryo-electron microscopy (cryo-EM) techniques will be used to analyze the assembly and uncoating intermediates of various viruses. The emphasis will be on the dsDNA tailed bacterial phages phi29 and T4 and the ssDNA icosahedral bacterial Microviridae phiX174 and alpha3. The objectives will be to determine not only the structures, but also the molecular mechanisms by which the assembly intermediates, including the DNA packaging process, are stabilized and then triggered for the subsequent steps in the viral morphogenesis. Similarly, the structural intermediates of viral disassembly during cellular infection will be examined. Cryo-EM will be used to determine the low resolution structures of the major assembly intermediates, the mature virus, and the DNA-emptied virus of phi29. X-ray crystallography will be used to determine the structures of most, if not all, of the 9 gene products that are required for the formation of the mature virus. This will allow the fitting of individual atomic resolution structures into the lower resolution EM densities. X-ray diffraction is being used to study components of the T4 phage baseplate. This baseplate, to which both the long and short tail fibers are attached, undergoes major conformational changes that initiate the recognition of cell surface receptors, the tail contraction, and the expulsion of the DNA from the viral head into the host cell. The available knowledge of the overall structure of this phage and its baseplate will be extended by cryo-EM. X-ray diffraction had been used earlier to study the mature phiX174 particle and an empty procapsid assembly byproduct. However, cryo-EM had to be used to capture the unstable, true procapsid assembly precursor. Currently, these studies are being repeated with the homologous phage alpha3. It is suspected that chimeric viruses of phiX174 and alpha3 may stabilize some of the assembly intermediates to permit crystallographic studies.The goal of the structural studies of viruses is to understand the molecular mechanisms of viral assembly and disassembly on cell infection. The assembly of a macromolecular structure proceeds along an ordered pathway and is accomplished by the switching of proteins between discrete conformations as they are added to the nascent assembly. Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. X-ray diffraction and cryo-electron microscopy techniques will be used to analyze the assembly and disassembly processes.

9986266Rossmann, m . x射线衍射和冷冻电子显微镜（cryo-EM）技术将用于分析各种病毒的组装和剥膜中间体。重点是dsDNA尾部噬菌体phi29和T4以及ssDNA二十面体细菌微病毒phiX174和alpha3。目标不仅是确定结构，还包括确定组装中间体（包括DNA包装过程）稳定的分子机制，然后触发病毒形态发生的后续步骤。同样，在细胞感染过程中，病毒分解的结构中间体也将被检查。Cryo-EM将用于确定phi29的主要组装中间体、成熟病毒和dna空化病毒的低分辨率结构。x射线晶体学将用于确定形成成熟病毒所需的9种基因产物中的大多数（如果不是全部的话）的结构。这将允许将单个原子分辨率结构拟合到较低分辨率的EM密度中。x射线衍射被用来研究T4噬菌体基板的成分。长尾和短尾纤维附着的基板经历了主要的构象变化，开始识别细胞表面受体，尾部收缩，并将DNA从病毒头部排出到宿主细胞中。通过低温电镜技术可以进一步了解噬菌体的整体结构及其基板。x射线衍射早前已用于研究成熟的phiX174粒子和空原衣壳组装副产物。然而，冷冻电镜必须用于捕获不稳定的，真正的原衣壳组装前体。目前，这些研究正在用同源噬菌体alpha3重复进行。我们怀疑phiX174和alpha3的嵌合病毒可能稳定了一些组装中间体，以便进行晶体学研究。病毒结构研究的目的是了解病毒在细胞感染过程中组装和拆卸的分子机制。大分子结构的组装沿着有序的途径进行，并通过将蛋白质添加到新生组装体中时在离散构象之间的切换来完成。支架蛋白通常在组装过程中起催化作用，就像分子伴侣一样。x射线衍射和低温电子显微镜技术将用于分析组装和拆卸过程。