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.

项目摘要 活细胞在空间和时间上有着复杂而精确的组织。了解蛋白质和 其他生物分子在体内形成功能网络是现代生物学的主要目标， 了解它们的正常功能和功能障碍。冷冻电子断层扫描（cryo-ET） 目前，这是唯一一种提供有关细胞内部生物结构的3D信息的成像技术， 分辨率优于~100，亚断层图像平均分辨率高达~30。然而，即使是30美分的分辨率 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3D 亚细胞结构的重建），以提高细胞冷冻电子显微镜（cryo-electron microscopy）的分辨率。 EM）至~15 <$，即，在这个水平上，蛋白质结构域可以被识别，并与已知的原子结构域进行比较。 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。虽然最终的TYGRESS平均值是 单粒子重建，具有比冷冻ET更高的分辨率，冷冻断层图像 作为粒子拾取和对齐的基本参考框架，这些步骤在其他情况下是不可能的， 复杂细胞标本的投影图像。我们将建立基础设施， 提供给广大的科学界。我们还将应用这一新工具来研究 细胞内三个重要的动态组件：（i）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） 这些蛋白质被转运穿过或插入内质网（ER）膜。泰格雷斯 将提供一个重要的和创新的桥梁之间的高分辨率结构的分离生物分子（x- 射线、NMR、单粒子冷冻-EM）和通过活细胞荧光成像的细胞内动力学可视化。