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: 9333405
• 负责人: Daniela Nicastro
• 金额: $ 35.64万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 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 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333405
• 关键词: Affect; Biological; Biological Models; Biology; Carrier Proteins; 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; Malignant Neoplasms; Membrane; Modernization; Molecular; Molecular Conformation; Noise; Organelles; Process; Protein translocation; Proteins; Protocols documentation; Radiation induced damage; Reporting; Research; Research Infrastructure; Resolution; Ribosomes; Roentgen Rays; Role; Rotavirus; Rotavirus Infections; Side; Signal Transduction; Software Tools; Specimen; Structure; Subcellular structure; Tertiary Protein Structure; Testing; Therapeutic; Thinness; Time; Tomogram; Translating; Virion; Virus; Virus Diseases; Work; cell motility; cellular imaging; ciliopathy; electron tomography; fluorescence imaging; graphical user interface; imaging approach; improved; in vivo; innovation; insight; macromolecular assembly; macromolecule; misfolded protein; movie; mutant; novel strategies; particle; protein E; protein transport; public health relevance; reconstruction; three dimensional structure; tomography; tool; treatment strategy

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.

描述(由申请人提供)：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，也是理解它们的正常功能和功能障碍的关键。冷冻电子断层成像(CRYO-ET)是目前唯一一种提供细胞内生物结构三维信息的成像技术，分辨率优于~100A，亚断层图像平均可达~30A。然而，即使是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)，以将细胞冷冻电子显微镜(Cryo-EM)的分辨率提高到~15A，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：冷冻-ET/亚断层图像平均和冷冻-EM单粒子重建。虽然最终的TYGRESS平均值是单粒子重建，具有比低温扫描更高的分辨率，但低温断层图像是粒子拾取和对准的必要参考框架，否则无法用于复杂细胞样本的投影图像。我们将建立基础设施，使这一新工具能够为广泛的科学界所用。我们还将利用这一新工具来研究细胞内三个重要的动态组装的结构和功能：(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)膜运输或插入内质网(ER)膜的分子机制。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之间提供一座重要的创新桥梁。