X-Ray Determination of Proteins and Viruses

9603571罗斯曼X射线衍射，使用结晶病毒，将是分析各种病毒的组装和剥离中间体的主要工具。然而，由于过渡中间体的短暂、不稳定的性质，通常不可能使其结晶。在这些情况下，X射线结晶学将通过低温电子显微镜得到增强。主要的重点将放在细菌病毒的研究上，例如单链X174和复杂得多的双链T4。更复杂、更大的dsDNA T4噬菌体有40多种结构蛋白。虽然不太可能通过X射线衍射来研究整个病毒，但研究单个蛋白质的原子结构是可能的。采用X射线衍射法和低温电子显微镜相结合的方法来确定各组分之间的相互作用。部分工作将是开发适当的技术，将低温电子显微镜与X射线衍射技术相结合，并检查缺乏完美二十面体对称性的粒子。目标将不仅是确定结构，而且还确定组装中间体被稳定并触发到随后的形态形成步骤的分子机制。同样，在细胞感染过程中病毒分解的结构中间体也将被检测。不同病毒系统的比较应该导致确定共同的策略和进化关系。这项研究的目的是阐明感染性病毒颗粒由其组成部分组装而成的机制。虽然这项工作致力于细菌病毒的研究，但病毒机制也可能被植物和动物病毒利用。还将研究病毒进入宿主期间的不同拆解过程。病毒组装通常需要帮助引导蛋白质折叠的支架蛋白质。因此，这些研究人员将是第一批观察到这些伴侣样蛋白与其底物复合的结构的研究人员之一。研究它们的瞬时中间体将需要开发结合X射线结晶学(可以分辨接近原子分辨率的特征)和冷冻电子显微镜(分辨率较低但缺乏制造晶体的要求)的技术。这些技术的结合很可能在结构生物学的未来发展中至关重要。

9603571 Rossmann X-ray diffraction, using crystallized viruses, will be the primary tool to analyze the assembly and uncoating intermediates of various viruses. Frequently, however, it is not possible to crystallize transient intermediates on account of their short-lived, unstable properties. In these cases, the X-ray crystallography will be augmented by cryo electron microscopy. The major emphasis will be on the study of bacterial viruses, such as single-stranded X174 and the far more complex double-stranded T4. The far more complex and far larger dsDNA T4 phage has over 40 structural proteins. While it is unlikely that it will ever be possible to study the whole virus by X-ray diffraction, it is possible to study the atomic structure of individual proteins. Combination of X-ray diffraction and cryo-EM is to be used to determine the interactions of components. Part of the work will be to develop suitable techniques for interfacing cryoelectron microscopy with X-ray diffraction techniques and also to examine particles that lack perfect icosahedral symmetry. The objectives will be to determine not only the structures, but also the molecular mechanisms by which the assembly intermediates are stabilized and triggered into the subsequent steps of morphogenesis. Similarly, the structural intermediates of viral disassembly during cellular infection are to be examined. Comparision of different viral systems should lead to the identification of common strategies and evolutionary relationships. The goal of the research is to elucidate the mechanisms by which infectious virus particles are assembled from their component parts. Although the work is devoted to the study of bacterial viruses, the viral mechanisms are also likely to be utilized by plant and animal viruses. The different process of disassembly during viral entry into a host will also be studied. Viral assembly often requires scaffolding proteins which help guide protein folding. Hence, the stud ies will be among the first to observe the structures of these chaperone-like proteins complexed with their substrates. The study of their transient intermediates will require the development of techniques to combine X-ray crystallography (which can resolve features near atomic resolution) and cryo-electron microscopy (which has poorer resolution but lacks the requirement of making crystals) Combination of these techniques is likely to be critical in the future development of structural biology.