Correlative cryo-microscopy: a new approach for characterizing the structure and

描述（由申请人提供）：细胞的大分子机器提出了跨越六个数量级的结构问题，从十分之一纳米（原子）到几十微米（细胞）。如此大范围的规模超出了任何一种技术的能力。因此，结构生物学家越来越多地转向使用混合方法。例如，x射线晶体学和低温电子显微镜的结合已被广泛用于在近原子水平上探测体外大型复合物（如病毒、核糖体、蛋白质体、伴侣蛋白、肌动蛋白丝和微管）的结构和力学。在这个项目中，我们将探索将这些第一代杂交技术、单分子光学显微镜和低温电子断层扫描技术联系起来的技术，以表征整个细胞背景下大分子复合物的结构和功能。将开发的技术包括使用光学成像方法来表征嵌入玻璃体冰的细胞中的大分子复合物的功能状态，并以非常高的精度（几十纳米）确定它们的位置，开发冷阶段，将允许光学位置映射转移到电子显微镜，开发计算工具来定位，识别，并在低温电子层析重建中确定大分子复合物的结构，并改进计算工具以使这些结构与传统体外方法获得的更高分辨率结构相匹配。在我们的初步研究中，我们将使用新技术来表征脊髓灰质炎病毒的细胞进入途径。选择该模型是因为其生物学相关性（非包膜病毒的细胞进入机制尚不清楚），并且因为几种相关的体外细胞进入中间体的结构已经已知。所开发的技术将普遍适用于广泛的大分子复合物。

DESCRIPTION (provided by applicant): The macromolecular machines of cells pose structural questions that span over six orders of magnitude in scale from the tenths of nanometers (atoms) to tens of microns (cells). This large range of scale exceeds the capabilities of any one technique. As a result, structural biologists have increasingly turned to the use of hybrid approaches. For example, the combination of x-ray crystallography and cryoelectron microscopy has been widely used to probe the structure and mechanics of large complexes (e.g. viruses, ribosomes, proteosomes, chaperones, actin filaments and microtubules) in vitro at the near atomic level. In this project we will explore technologies to link these first generation hybrid techniques, single molecule optical microscopy, and cryoelectron tomography to characterize the structure and function of macromolecular complexes in the context of the whole cell. The technologies that will be developed include the use of optical imaging methods to characterize the functional state of macromolecular complexes in cells imbedded in vitreous ice and determine their location with very high precision (tens of nm), development of a cold stage that will permit the optical positional mapping to be transferred to the electron microscope, development of computational tools to locate, identify, and determine the structures of macromolecular complexes in cryoelectron tomographic reconstructions, and refinement of computational tools to match these structures with higher resolution structures derived from conventional in vitro methods. In our initial studies we will use the new technologies to characterize the cell entry pathway of poliovirus. This model was chosen because of its biological relevance (the cell entry mechanisms of nonenveloped viruses are poorly understood) and because structures of several relevant cell entry intermediates in vitro are already known. The techniques developed will be generally applicable to a wide range of macromolecular complexes. In this project will develop technologies that will combine fluorescence optical microscopy and cryoelectron tomography to characterize the structure and function of intracellular macromolecular assemblies at the molecular level in situ. The technology will be relevant to a wide range of intracellular structures, and will add significantly to our basic understanding of the structure and function of "cellular machines."