CRYO CORE

完整细胞的冷冻电子断层扫描可能提供一个全球背景，以统一来自 结晶学/核磁共振和单分子方法，使确定空间 细胞中关键蛋白质和复合体的排列，具有能够跟随的令人兴奋的前景 信令事件期间这些分布的空间和时间变化288。尽管大家都知道 这种方法的巨大潜力，有几个局限性阻碍了它在细胞和 到目前为止的结构生物学。首先，细胞的厚度，特别是哺乳动物细胞(&gt；1um)，限制了 可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(单元格内部拥挤)，这通常会导致对 断层扫描体积内的密度和感兴趣的空间排列的描绘 分子困难；第三，鉴于低剂量成像的性质，其中辐射对生物造成损害 样本需要最小化，感兴趣区域的地点通常在数据之前是未知的 收购。我们提出了以下具体目标来开发和实现低温电子工具 克服这些技术障碍并使冷冻电子断层成像成为标准的断层扫描 检测艾滋病毒-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.