Resolution Improvement for Cellular CryoEM to study Dynamic Assemblies in Cells

描述（由申请人提供）：活细胞在空间和时间上具有复杂且通常精确的组织。了解蛋白质和其他生物分子如何在体内形成功能网络是现代生物学的主要目标，对于理解它们的正常功能和功能障碍至关重要。低温电子断层扫描（cryo-ET）是目前唯一能提供细胞内生物结构三维信息的成像技术，分辨率优于~100 a，亚层析成像平均分辨率可达~30 a。然而，即使30a的分辨率也不能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”（断层扫描引导的亚细胞结构3D重建），以提高细胞冷冻电子显微镜（cryo- EM）的分辨率至~15 a，即可以识别蛋白质结构域并与已知原子结构进行比较的水平。我们的方法结合了两种成像技术的互补优势：低温ET/亚层析成像平均和低温em单粒子重建。虽然最终的TYGRESS平均值是单粒子重建，具有比cryo-ET更高的分辨率，但cryo- tomography可以作为粒子拾取和对准的基本参考框架，这些步骤对于复杂细胞标本的投影图像来说是不可能的。我们将建立基础设施，使这个新工具能够被广泛的科学界使用。我们还将应用这个新工具来研究细胞内三个重要的动态组件的结构和功能：(i)纤毛和鞭毛的分子组织和功能，（ii）轮状病毒进入和宿主细胞感染的“分子电影”，以及（iii）蛋白质通过内质网（ER）膜运输或插入的分子机制。TYGRESS将在分离生物分子的高分辨率结构（x射线、核磁共振、单粒子低温电镜）和活细胞荧光成像的细胞内动力学可视化之间提供必不可少的创新桥梁。

DESCRIPTION (provided by applicant): 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 A, and up to ~30 A with subtomogram averaging. However, even 30 A 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 A, 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) he 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.