Resolution Improvement for Cellular CryoEM to study Dynamic Assemblies in Cells

项目摘要 活细胞在空间和时间上有一个复杂的、往往是精确的组织。了解蛋白质和蛋白质如何 其他生物分子在体内形成功能网络是现代生物学的一个主要目标，并且对 了解他们的正常功能和功能障碍。冷冻电子断层扫描(CRYO-ET)是 目前唯一一种提供细胞内生物结构3D信息的成像技术 分辨率优于~100？，亚断层图像平均分辨率可达~30？然而，即使是30%的分辨率 没有提供识别大多数蛋白质或可视化它们之间相互作用所需的细节。 因此，我们将开发一种新的混合方法和软件工具--TYGRESS(层析制导3D 亚细胞结构的重建)，以提高细胞低温电子显微镜(低温电子显微镜)的分辨率。 Em)到~15°，即蛋白质结构域可以被识别并与已知原子进行比较的水平 结构。我们的方法结合了两种成像技术的互补优势：冷冻- ET/亚断层图像平均和冷冻-EM单粒子重建。而最终的TYGRESS平均值是 单粒子重建的好处是分辨率比冷冻、冷冻断层成像更高 用作粒子拾取和对齐的基本参考坐标系，否则无法实现以下步骤 复杂细胞标本的投影图像。我们将建立基础设施，使这一新工具 可供广泛的科学界使用。我们还将应用这一新工具来研究细胞的结构和功能 细胞内三个重要的动态组装：(I)纤毛和纤毛的分子组织和功能 鞭毛，(Ii)轮状病毒进入和宿主细胞感染的“分子电影”，以及(Iii)通过 哪些蛋白质通过内质网(ER)膜运输或插入内质网(ER)膜。TYGRESS 将在分离生物分子的高分辨率结构(x- 射线、核磁共振、单颗粒低温电子显微镜)和通过活细胞荧光成像显示细胞内动力学。

Project Summary Living cells have a complex and often precise organization in space and time. Understanding how proteins and other biomolecules form functional networks in vivo is a major goal of modern biology, and paramount to understanding both their normal functions as well as dysfunctions. Cryo-electron tomography (cryo-ET) is currently the only imaging technique that provides 3D information about biological structures inside cells, at a resolution better than ~100 ¿, and up to ~30 ¿ with subtomogram averaging. However, even 30 ¿ resolution does not provide the detail needed to identify most proteins or visualize their interactions with one another. Therefore, we will develop a new hybrid approach and software tool, "TYGRESS" (Tomography Guided 3D Reconstruction of Subcellular Structures), to improve the resolution of cellular cryo-electron microscopy (cryo- EM) to ~15 ¿, i.e., a level at which protein domains can be recognized and compared to known atomic structures. Our approach combines the complementary strengths of two imaging techniques: cryo- ET/subtomogram averaging and cryo-EM single-particle reconstruction. While the final TYGRESS average is a single-particle reconstruction with the benefit of a higher resolution than possible by cryo-ET, cryo-tomograms serve as essential reference frames for particle picking and alignment, steps that are not otherwise possible for projection images of complex cellular specimens. We will build the infrastructure to make this new tool available to a wide scientific community. We will also apply this new tool to study the structure and function of three important and dynamic assemblies inside cells: (i) The molecular organization and function of cilia and flagella, (ii) a "molecular movie" of rotavirus entry and host cell infection, and (iii) the molecular mechanisms by which proteins are transported across or inserted into the endoplasmic reticulum (ER) membrane. TYGRESS will provide an essential and innovative bridge between high-resolution structures of isolated biomolecules (x- ray, NMR, single-particle cryo-EM) and visualization of intracellular dynamics by live-cell fluorescence imaging.