X-Ray Determination of Proteins and Viruses

9603571 Rossmann X射线衍射，使用结晶病毒，将是主要的工具来分析各种病毒的组装和脱壳中间体。 然而，由于短暂的中间体存在时间短，性质不稳定，通常不可能使其结晶。 在这些情况下，X射线晶体学将通过低温电子显微镜来增强。 主要重点将放在细菌病毒的研究上，如单链X174和更复杂的双链T4。 更复杂和更大的dsDNA T4噬菌体具有超过40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。X射线衍射和冷冻EM的组合将用于确定组分的相互作用。 这项工作的一部分将是开发合适的技术，使低温电子显微镜与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颗粒。 我们的目标将是确定不仅是结构，但也组装中间体的分子机制是稳定和触发到随后的步骤形态发生。 同样，在细胞感染过程中病毒分解的结构中间体也要进行检查。 不同病毒系统的比较应该导致共同策略和进化关系的识别。 这项研究的目的是阐明感染性病毒颗粒从其组成部分组装的机制。 虽然这项工作致力于研究细菌病毒，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分解过程。 病毒装配通常需要支架蛋白来帮助引导蛋白质折叠。 因此，这些研究将是第一批观察到这些分子伴侣样蛋白与其底物复合的结构的研究。 对它们的瞬态中间体的研究将需要发展结合联合收割机X射线晶体学（可以分辨接近原子分辨率的特征）和低温电子显微镜（分辨率较差，但缺乏制造晶体的要求）的技术。这些技术的结合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.