X-ray Determinations of Proteins and Viruses

1963年这个项目最初的标题是“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，从上世纪90年代开始，随着第一个病毒结构的成功确定和晶体学作为自动化工具的建立，该项目被定向用于支持感染和杀死细菌的病毒（“噬菌体”）的研究。在当前项目期间，有一个转变，不仅包括噬菌体的分析，而且还包括通过冷冻电子断层扫描（cryoET）分析多形性病毒的新工具的开发。x射线衍射和低温电子显微镜（cryoEM）技术，结合分子生物学和蛋白质化学，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上，无尾噬菌体phi X174；双链DNA （dsDNA）上为短尾噬菌体C1；在小的，dsDNA，尾部噬菌体phi 29；以及大而复杂的dsDNA噬菌体phi KZ。这个项目的部分重点将是扩展先前的一些低温电镜重建，使单个氨基酸的可视化成为可能，这些氨基酸决定了病毒成分的化学性质，从而决定了病毒成分的功能。此外，已经开始了对不完全服从二十面体对称的脂质包膜病毒的冷冻层析成像的解释研究。这类病毒要么形状过于笨拙，无法形成足够的颗粒间接触以实现结晶的包装组织，要么是多形性的（许多不同的形状），没有病毒粒子具有完全相同的形状或形式。必要时，冷冻电镜和冷冻et将通过病毒蛋白成分的晶体结构进行扩增。拟原子分辨率结构可以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较大片段的冷冻电镜或冷冻电镜图像中来获得。通过将结构结果与组装中间体、基因组包装和宿主识别的突变和其他研究相结合，希望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，因此在噬菌体研究中获得的信息对所有病毒的研究都有帮助。更广泛的影响在NSF支持该项目的过去五年中发表的论文的共同作者包括一名本科生，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。这三位研究生现在都获得了博士学位，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利用了一个学期的晶体学（由PI教授）和电子显微镜课程。在这个项目的未来五年里，可能会有类似的统计数据。自1963年以来，已有130多名博士后和30多名研究生受益于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.