Structural basis of bacteriophage T7 assembly and DNA packaging

描述（由申请人提供）：T7是研究最多的噬菌体系统之一，具有丰富的遗传和生化信息。与其他尾部dsDNA噬菌体类似，T7的形态发生途径始于无dna的空原衣壳，该原衣壳通过支架蛋白辅助组装形成。在成熟过程中，原衣壳发生了大规模的构象变化，并扩展到衣壳II状态，这是由DNA通过门户顶点包装引发的。只有在DNA包装和尾部组装完成后，才会形成具有传染性的颗粒。尽管包括T7在内的许多噬菌体系统已经对噬菌体形态发生进行了广泛的研究，但其机制仍有许多问题有待解决。在对T7衣壳结构有了初步的了解后，我们建议开展更系统的研究来阐明T7组装、成熟和DNA包装的结构基础。我们的目标是解决衣壳壳，支架蛋白，门户复合体，内部圆柱形核心，尾部和DNA包装的结构。这些结构将在形态发生途径中的每个衣壳状态下进行检查，以探索它们在成熟过程中的作用。最先进的低温电子显微镜和计算机三维重建技术将用于完成这些任务。我们将使用流行的图像处理软件EMAN， PI是其中的共同开发者，通过施加二十面体对称来确定衣壳外壳结构到7-8 a或更好的分辨率。我们还将进一步开发图像处理算法，以推动传染性噬菌体颗粒的4-5 A分辨率结构。我们最近对EMAN进行了扩展，以允许对称匹配结构的解决，将用于以15 A或更高的分辨率确定完整衣壳中的支架蛋白，门户复合体，圆柱核心和尾部的结构。这些蛋白质不具有二十面体对称性，不能使用依赖二十面体平均的经典重建方法来解决。该方法将进一步发展，以允许直接跟踪dsDNA链和可视化的dsDNA包装。T7结构将为理解尾部dsDNA噬菌体以及相关人类疱疹病毒的组装、成熟和基因组包装的基本过程奠定基础。

DESCRIPTION (provided by applicant): T7 is one of the most studied bacteriophage systems for which a wealth of genetic and biochemical information is known. Similar to other tailed dsDNA phages, the morphogenesis pathway of T7 starts with a DNA-free empty procapsid that is formed through scaffolding protein-assisted assembly. The procapsid undergoes large-scale conformational changes and expands into the capsid II state during the maturation process, which is triggered by DNA packaging through the portal vertex. An infectious particle is formed only after the completion of DNA packaging and tail assembly on the portal vertex. Although phage morphogenesis has been extensively studied for numerous phage systems, including T7, many questions remain to be answered to understand its mechanism. Having been able to obtain some preliminary results on the structures of the T7 capsids, we propose to carry out more systematic studies to elucidate the structural basis of T7 assembly, maturation and DNA packaging. We aim to solve the structures of the capsid shell, the scaffolding proteins, the portal complex, the internal cylindrical core, the tail and the DNA packing. These structures will be examined at each of their capsid states in the morphogenesis pathway to probe their roles in the maturation process. State-of-the-art cryo-electron microscopes and computer 3-D reconstruction techniques will be used to accomplish these tasks. We will use the popular image processing software EMAN, of which the PI is a co-developer, to determine the capsid shell structures to 7-8 A or better resolution by imposing icosahedral symmetry. We will also further develop the image processing algorithms to push for 4-5 A resolution structures for the infectious phage particles. Our recent extension of EMAN to allow the solution of structures of symmetry matches will be used to determine the structures of the scaffolding proteins, portal complex, cylindrical core and tail in the intact capsids at 15 A resolution or better. These proteins do not have icosahedral symmetry and cannot be resolved using classic reconstruction methods that rely on icosahedral averaging. This method will be further developed to allow direct tracing of the dsDNA strands and visualization of dsDNA packing. The T7 structures will form the foundation for understanding the basic processes of assembly, maturation and genome packaging of not only the tailed dsDNA phages, but also the related human herpes viruses.