Resolution Improvement for Cellular CryoEM to study Dynamic Assemblies in Cells

提高细胞冷冻电镜的分辨率以研究细胞中的动态组装

• 批准号: 8749864
• 负责人: Daniela Nicastro
• 金额: $ 37.67万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8749864
• 关键词: Affect; Biological; Biological Models; Biology; Cell membrane; Cell physiology; Cells; Cellular Structures; Cilia; Classification; Communities; Complex; Computer software; Cryoelectron Microscopy; Cystic Fibrosis; Data; Defect; Development; Diagnostic; Disease; Documentation; Dose; Educational Status; Electrons; Elements; Endoplasmic Reticulum; Environment; Esthesia; Flagella; Functional disorder; Goals; Human; Hybrids; Image; Imagery; Imaging Device; Imaging Techniques; In Situ; Infection; Internet; Lead; Left; Life; Malignant Neoplasms; Membrane; Molecular; Molecular Conformation; Noise; Organelles; Process; Protein translocation; Proteins; Protocols documentation; Radiation; Reporting; Research; Research Infrastructure; Resolution; Ribosomes; Role; Rotavirus; Rotavirus Infections; Side; Signal Transduction; Software Tools; Specimen; Staging; Structure; Subcellular structure; Tertiary Protein Structure; Testing; Therapeutic; Time; Tomogram; Translating; Virion; Virus; Virus Diseases; Work; cell motility; cellular imaging; ciliopathy; electron tomography; fluorescence imaging; graphical user interface; improved; in vivo; innovation; insight; macromolecular assembly; macromolecule; movie; mutant; novel strategies; particle; protein misfolding; protein transport; public health relevance; reconstruction; three dimensional structure; tomography; tool; treatment strategy

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.

项目概要 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，对于 了解它们的正常功能和功能障碍。冷冻电子断层扫描 (cryo-ET) 是 目前唯一能够提供细胞内生物结构 3D 信息的成像技术 分辨率优于约 100 ¿，并且采用次断层扫描平均时高达约 30 ¿然而，即使是 30 ¿ 没有提供识别大多数蛋白质或可视化它们彼此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3D 重建亚细胞结构），提高细胞冷冻电子显微镜（cryo- EM) 至 ~15 ¿，即可以识别蛋白质结构域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/亚断层扫描平均和冷冻电镜单粒子重建。虽然最终的 TYGRESS 平均值是 单粒子重建具有比冷冻电子断层扫描、冷冻断层扫描更高的分辨率 作为粒子拾取和对齐的基本参考系，这些步骤是其他方法不可能实现的 复杂细胞样本的投影图像。我们将构建基础设施来制造这个新工具 可供广大科学界使用。我们还将应用这个新工具来研究其结构和功能 细胞内三个重要且动态的组装：（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) 的分子机制 这些蛋白质被转运穿过或插入内质网（ER）膜。泰格雷斯 将在分离生物分子的高分辨率结构（x- 射线、核磁共振、单粒子冷冻电镜）以及通过活细胞荧光成像实现细胞内动力学可视化。