The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.

中文翻译：

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使用TYGRESS在纳米分辨率下进行原位结构测定。

由拥挤的细胞环境的低温电子断层图（cryo-ET）计算出的子图平均分辨率通常受到限制，这是由于子图的信号丢失和未对准所致。相比之下，单粒子低温电子显微镜（SP-cryo-EM）通常可以达到分离出的复合物的近原子分辨率。我们报告了一种称为“层析成像引导的亚细胞结构3D重建”（TYGRESS）的方法，该方法是cryo-ET和SP-cryo-EM的混合体，能够在拥挤的细胞环境中实现接近纳米级的复合物分辨率。TYGRESS结合了SP-cryo-EM（具有良好信噪比和对比度的图像，以及最小的辐射损伤）和子图平均（复杂样品中大分子的三维排列）的优势。使用TYGRESS，我们确定了完整睫状轴突的结构，分辨率高达12Å。这些结果揭示了仅cryo-ET看不见的许多结构细节。TYGRESS通常适用于适合于子图平均的细胞复合物。