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（cryoEM）技术，结合分子生物学和蛋白质化学，将是用于分析各种细菌病毒的生命周期的主要工具。重点将放在小而简单的双链DNA（dsDNA）尾噬菌体phi29上，在大而复杂的dsDNA噬菌体T4和甚至更大的phiKZ上，以及在无尾二十面体ssDNA噬菌体phiX174上。目标将是确定不仅是结构，而且组装中间体稳定的分子机制，DNA如何包装到空的预组装前体中，在病毒形态发生过程中每个步骤如何触发下一个步骤，并研究病毒感染宿主细胞时发生的巨大构象变化。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并不容易通过单独研究动物病毒来确定。许多细菌病毒的形状过于笨拙，无法形成足够紧凑的包装组织，并有足够的颗粒间接触，使结晶成为可能。此外，这些病毒具有许多纤维传感器附件，这些附件是柔性的并且在方向上可变，这将使得结晶化是不可能的。尽管目前病毒的cryoEM研究很少超过10埃分辨率，但这些研究可以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来增强。通过将单个晶体结构拟合到病毒粒子、组装中间体或天然病毒的较大片段的cryoEM图像中，可以获得伪原子分辨率的结构。更广泛的影响：该项目将涉及本科生、研究生和博士后研究员。PI一直积极指导妇女以及代表性不足的少数民族研究人员。此外，这项研究将纳入教学活动，包括结晶学课程。与过去一样，该项目将涉及新技术的开发，例如结合电子显微镜和晶体学。这些技术将有助于科学界研究大型复合体的结构生物学。

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.