X-ray Determinations of Proteins and Viruses

智慧的价值这个项目1963年最初的标题，“蛋白质和病毒的X射线测定”，今天仍然适用。在60年代和70年代，该项目主要支持结构研究技术的发展。然而，从90年代开始，随着第一批病毒结构的成功确定和作为自动化工具的结晶学的建立，该项目旨在支持感染并能够杀死细菌的病毒(“噬菌体”)的研究。在目前的项目期间，不仅包括对噬菌体的分析，而且还包括开发新的工具，用于通过冷冻电子断层扫描(低温电子断层扫描)分析多形性病毒。X射线衍射和低温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。重点将放在小的单链DNA，无尾噬菌体Phi x174；双链DNA(DsDNA)，短尾噬菌体C1；小的dsDNA，尾部噬菌体Phi 29；以及大而复杂的dsDNA噬菌体Phi KZ。该项目的部分重点将是扩展以前的一些低温EM重建，以使决定化学从而决定病毒组件功能的单个氨基酸的可视化成为可能。此外，已经开始研究对不符合确切二十面体对称性的脂膜病毒的冷冻断层图像的解释。这种病毒要么形状太笨拙，无法形成具有足够颗粒间接触的包装组织，使结晶可行，要么是多形性的(许多不同的形状)，没有具有完全相同形状或形式的病毒粒子。在必要的情况下，病毒蛋白成分的晶体结构将增强低温EM和低温ET。通过将单个晶体结构与病毒粒子、组装中间体或更大的天然病毒片段的冷冻电子显微镜或冷冻电子显微镜图像进行匹配，可以获得伪原子分辨结构。通过将结构结果与突变和其他对组装中间体、基因组包装和宿主识别的研究相结合，希望能探索这些生物过程的机制。由于噬菌体和病毒都需要组装和宿主识别等功能，从噬菌体研究中获得的信息可能对所有病毒的研究都有帮助。布罗德影响过去五年NSF对该项目的支持发表的论文的合著者包括一名本科生、三名研究生、十一名博士后和三名技术人员。其中7人是女性，他们的种族背景包括1名西班牙裔、3名中国人、1名日本人、3名俄罗斯人、1名韩国人、1名加拿大人和4名美国人。这三名研究生现在都已经获得了博士学位，并继续进行博士后培训。其中两名学生现在是主要大学的助理教授。所有学生和大多数博士后都参加了为期一学期的结晶学(由PI教授)和电子显微镜课程。类似的统计数据可能会在该项目的未来五年内公布。自1963年以来，已有130多名博士后和30名研究生受益于NSF的持续支持。这些学生和博士后中的许多人后来成为了国际公认的结构生物学家。

Intellectual MeritThe original 1963 title of this project, "X-ray Determinations of Proteins and Viruses," is still apt today. In the 60s and 70s the project supported primarily the development of technology for structural studies. However, starting in the 90s, with the successful determination of the first virus structures and the establishment of crystallography as an automated tool, this project was directed to support the study of viruses that infect and can kill bacteria ("bacteriophages"). In the period of the current project there is a shift back to include not only analyses of bacteriophages, but also the development of new tools for the analysis of pleomorphic viruses by means of cryo-electron tomography (cryoET). 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, single-stranded DNA, tailless bacteriophage phi X174; on the double-stranded DNA (dsDNA), short-tailed phage C1; on the small, dsDNA, tailed phage phi 29; as well as on the large and complex dsDNA phage phi KZ. Part of the emphasis of this project will be to extend some of the previous cryoEM reconstructions to make possible visualization of individual amino acids that determine chemistry and, hence, the function of the viral components. In addition, studies have been initiated on the interpretation of cryo-tomograms of lipid enveloped viruses that do not obey exact icosahedral symmetry. Such viruses are either too awkwardly shaped to achieve a packing organization that has sufficient inter-particle contacts to make crystallization feasible, or are pleomorphic (many different shapes) with no virion having exactly the same shape or form. Where necessary, cryoEM and cryoET will be augmented by crystal structures of viral protein components. Pseudo-atomic resolution structures can be obtained by fitting the individual crystal structures into the cryoEM or cryoET images of the virion, assembly intermediates or larger fragments of the native virus. By combining structural results with mutational and other studies of assembly intermediates, genome packaging and host recognition, the hope is to probe the mechanisms of these biological processes. As functions such as assembly and host recognition that are required by both bacteriophages as well as by viruses, information gained in the study of bacteriophages can be helpful in the study of all viruses.Broader ImpactThe co-authors of the papers published over the previous five years of NSF support for this project include one undergraduate, three graduate students, eleven post-docs and three technicians. Of these, seven were women and their ethnic backgrounds included one Hispanic, three Chinese, one Japanese, three Russian, one Korean, one Canadian and four US Americans. All three of the graduate students have now received their Ph.D. degrees and went on to do postdoctoral training. Two of these students are now assistant professors at major universities. All students and most post-docs took advantage of a semester long courses in crystallography (taught by the PI) and electron microscopy. Similar statistics are likely for the next five years of this project. Since 1963 more than 130 post-docs and 30 graduate students have benefited from the continuing NSF support. Many of these students and post-docs have subsequently become internationally recognized structural biologists.