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

相关冷冻显微镜：一种表征结构和特征的新方法

基本信息

批准号：
7664279
负责人：
JAMES M HOGLE
金额：
$ 33.9万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2012-07-31
项目状态：
已结题

项目摘要

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."

描述（由申请人提供）：细胞的大分子机器提出结构性问题，这些问题的规模超过六个数量级，从十分之一的纳米（原子）到数十微米（细胞）。这种较大的规模超过了任何一种技术的功能。结果，结构生物学家越来越多地转向使用混合方法。例如，X射线晶体学和冷冻电子显微镜的组合已被广泛用于探测大型复合物（例如病毒，核糖体，蛋白质体，伴侣，伴侣蛋白，肌动蛋白细丝和微管）的结构和力学。在这个项目中，我们将探索技术，以将这些第一代混合技术，单分子光学显微镜和冷冻电子断层扫描联系起来，以表征整个细胞背景下大分子复合物的结构和功能。将要开发的技术包括使用光学成像方法来表征在玻璃体中嵌入的细胞中大分子复合物的功能状态，并以非常高的精确度（NM的数十个）来确定其位置，开发寒冷阶段，该阶段将允许光学位置映射，以将其转移到计算机上，并确定计算的工具，并确定识别物的位置，并确定识别，并确定，并确定，并确定了识别，并确定，并确定了识别，并确定了该位置，并将其定位，并允许将其定位，并允许将其定位，并允许将其定位，并可以将其转移到位置，并允许将其定位，并允许将其转移到较冷的位置映射，并允许将其转移到较冷的位置映射，并允许将其转移到较冷的位置映射，并可以将其转移到较冷的位置映射。冷冻电子层析成像重建和计算工具的改进，以使这些结构与传统体外方法得出的较高分辨率结构相匹配。在我们的最初研究中，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。之所以选择该模型，是因为其生物学相关性（未开发病毒的细胞进入机制知之甚少），并且由于已经知道了几种相关细胞进入中间体的结构。开发的技术通常适用于广泛的大分子复合物。在该项目中，将开发技术，将结合荧光光学显微镜和冷冻电子断层扫描，以表征细胞内大分子分子组件在原位分子水平的结构和功能。该技术将与各种细胞内结构有关，并将大大增加我们对“蜂窝机”结构和功能的基本理解。