CRYO CORE

完整细胞的冷冻电子断层扫描可能提供一个全球范围内统一所有结构数据， 晶体学/NMR和单分子方法，使得有可能确定空间 细胞中关键蛋白质和复合物的排列，以及能够遵循的令人兴奋的前景 信号事件期间这些分布的空间和时间变化288.尽管公认的 尽管这种方法具有巨大的潜力，但一些限制阻碍了其在细胞和 结构生物学至今。首先，细胞，特别是哺乳动物细胞（>1 μ m）的厚度限制了细胞的生长。 可以从全细胞断层图像中恢复的有用信息量;第二，细胞断层图像 包含过多的信息（细胞内部拥挤），这通常会导致对 在断层扫描体积内的密度和感兴趣的空间排列的描绘 第三，考虑到低剂量成像的性质，其中辐射对生物分子的损伤是非常大的。 样本需要最小化，感兴趣区域的位置通常在数据之前是未知的。 采集我们提出了以下具体目标，以开发和实施用于低温电子 断层扫描克服这些技术障碍，使低温电子断层扫描成为标准 检测HIV-宿主细胞相互作用的技术。

Cryo-electron tomography of intact cells is likely to provide a global context to unify all structural data from crystallography/NMR and single molecule approaches, making it possible to determine the spatial arrangements of key proteins and complexes in the cell, with the exciting prospect of being able to follow spatial and temporal changes in these distributions during signaling events288. Despite the acknowledged immense potential of this methodology, several limitations have hindered its broader application in cellular and structural biology up to now. First, the thickness of cells, mammalian cells in particular (>1 um), limits the amount of useful information that can be recovered from whole cell tomograms; second, cellular tomograms contain an overabundance of information (interiors of cells are crowded), which often renders interpretation of densities within the tomographic volume and delineation of the spatial arrangement of the interesting molecules difficult; and third, given the nature of low dose imaging in which radiation damage to the biological specimen needs to be minimized, the locale of the region of interest is generally not known prior to data acquisition. We propose the following specific aims to develop and implement tools for cryo-electron tomography to overcome these technological barriers and enable cryo-electron tomography as a standard technology for examining HIV-host cell interactions.