X-ray Determination of Proteins and Viruses

9986266Rossmann、M.X-射线衍射和低温电子显微镜(Cryo-EM)技术将用于分析各种病毒的组装和剥离中间体。重点将放在dsDNA尾部噬菌体phi29和T4以及ssDNA二十面体细菌微病毒科phiX174和alpha3上。其目标将不仅是确定结构，而且还确定组装中间产物(包括DNA包装过程)被稳定并随后被触发以用于病毒形态发生的后续步骤的分子机制。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。冷冻-电子显微镜将用于确定主要组装中间体、成熟病毒和DNA清除的phi29病毒的低分辨结构。X射线结晶学将用于确定成熟病毒形成所需的9个基因产物中的大多数(如果不是全部)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。这种基板上既有长尾纤维，也有短尾纤维，它经历了重大的构象变化，启动了细胞表面受体的识别、尾部收缩以及DNA从病毒头部排出到宿主细胞。有关该噬菌体及其底板整体结构的现有知识将通过冷冻-EM得到扩展。X-射线衍射法曾被用来研究成熟的phiX174颗粒和一个空的Proapsid组装副产物。然而，必须使用冷冻EM来捕获不稳定的、真正的普罗卡西德组装前体。目前，这些研究正在与同源噬菌体alpha3重复。据推测，phiX174和alpha3嵌合病毒可能稳定了一些组装中间体，从而进行了结晶学研究。病毒结构研究的目标是了解病毒组装和拆解在细胞感染中的分子机制。大分子结构的组装沿着有序的路径进行，当蛋白质加入到新生的组装中时，通过蛋白质在离散构象之间的转换来完成。支架蛋白通常在组装过程中起催化作用，很像分子伴侣。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。

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.