X-ray Determination of Proteins and Viruses

在这个项目中，X射线衍射和冷冻电子显微镜技术与分子生物学和蛋白质化学相结合，将成为分析各种细菌病毒生命周期的主要工具。重点将放在小而简单的双链DNA(DsDNA)尾部噬菌体phi29，大而复杂的dsDNA噬菌体T4，甚至更大的phiKZ，以及无尾的二十面体单链DNA噬菌体phiX174。其目标将不仅是确定组装中间体的结构，还包括稳定组装中间体的分子机制，DNA如何包装到空的预组装探针中，病毒形态发生期间每一步如何触发下一步，以及研究病毒感染宿主细胞时发生的巨大构象变化。对细菌和动物病毒系统的比较可能会认识到共同的策略和进化起源，而这些策略和进化起源不是仅仅通过动物病毒的研究就能轻易确定的。许多细菌病毒的形状太笨拙，无法实现足够紧凑的包装组织，以及足够的颗粒间接触，从而使结晶成为可能。此外，这些病毒有许多纤维传感器附着物，这些附着物是灵活的，方向可变，这将使结晶变得不可能。尽管目前对病毒的低温电子显微镜研究很少超过10埃分辨率，但通过单独研究在合适的原核或真核系统中表达的病毒组分的晶体结构，这些研究可以得到加强。然后，通过将单个晶体结构与病毒粒子、组装中间体或更大的本地病毒片段的低温电子显微镜图像进行匹配，可以获得伪原子分辨结构。广泛的影响：该项目将涉及本科生、研究生和博士后研究员。国际和平协会一直在积极指导妇女以及代表性不足的少数族裔研究人员。此外，这项研究将融入教学活动，包括一门关于结晶学的课程。与过去一样，该项目将涉及新技术的开发，如结合电子显微镜和结晶学。这些技术将对从事大型复合体结构生物学工作的科学界有用。

In this project, X-ray diffraction and cryo-electron microscopy (cryoEM) techniques, in conjunction with molecular biology and protein chemistry, will be the primary tools used to analyze the life cycle of various bacterial viruses. The emphasis will be on the small and simple double-stranded DNA (dsDNA) tailed bacteriophage phi29, on the large and complex dsDNA bacteriophages T4 and even larger phiKZ, as well as on the tailless icosahedral ssDNA bacteriophage phiX174. The objectives will be to determine not only the structures, but also the molecular mechanisms by which the assembly intermediates are stabilized, how the DNA is packaged into the empty pre-assembled proheads, how each step triggers the next during viral morphogenesis, and to study the huge conformational changes that occur when the virus infects a host cell. Comparison of bacterial and animal viral systems is likely to recognize common strategies and evolutionary origins that are not as easily determined by the study of animal viruses alone. Many bacterial viruses are too awkwardly shaped to achieve a packing organization that is compact enough, with sufficient interparticle contacts, to make crystallization feasible. In addition, these viruses have numerous fibrous sensor attachments that are flexible and variable in orientation that would make crystallization impossible. Although cryoEM investigations of viruses seldom extend beyond 10-angstrom resolution at this time, these studies can be augmented by separately studying the crystal structures of the viral components expressed in suitable prokaryotic or eukaryotic systems. A pseudo-atomic resolution structure can then be obtained by fitting the individual crystal structures into the cryoEM images of the virion, assembly intermediates, or larger fragments of the native virus.Broader Impacts: The project will involve undergraduates, graduate students, and postdoctoral fellows. The PI has been active in mentoring women as well as underrepresented minority researchers. In addition, the research will be integrated in the teaching activities, including a course on crystallography. As in the past, the project will involve development of new techniques, such as combining electron microscopy and crystallography. Such techniques will be useful to the scientific community working on the structural biology of large complexes.