High-Resolution CryoEM Reconstruction of Large Complexes

大型复合物的高分辨率冷冻电镜重建

• 批准号: 8501521
• 负责人: Z Hong ZHOU
• 金额: $ 27.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8501521
• 关键词: Abbreviations; Achievement; Alanine; Algorithms; Amino Acids; Amyloid Fibrils; Aquareoviruses; Area; Astigmatism; Attention; Binding; Biochemistry; Biological; Biological Process; Biomedical Research; Bluetongue virus; Cellular biology; Communities; Complement; Complement Factor B; Complex; Computer software; Computers; Coupled; Cryoelectron Microscopy; Crystallization; Cytoplasmic Polyhedrosis Viruses; Cytoskeletal Filaments; Data; Devices; Disease; Drug Design; Electron Beam; Electrostatics; Emerging Technologies; Enzymatic Biochemistry; Fc Receptor; Film; Flagella; Fourier Transform; Frequencies; Funding; Goals; Hepatitis B Virus; Herpesvirus 1; Image; Imagery; Individual; Intervention; Maps; Measurement; Methods; Mission; Modeling; Molecular Genetics; Molecular Medicine; Nature; Noise; Occupations; Outcome; Pathology; Performance; Pharmaceutical Preparations; Phase; Pilum; Positioning Attribute; Process; Proteins; Publishing; Research Personnel; Residual state; Resolution; Role; Sampling; Shapes; Side; Signal Transduction; Solutions; Speed; Structure; System; Techniques; Technology; Testing; Time; Tobacco Mosaic Virus; United States National Institutes of Health; Validation; Vertebral column; Virus; Work; X-Ray Crystallography; base; computer cluster; computerized data processing; computerized tools; cost effective; covalent bond; density; design; disease mechanisms study; falls; graphical user interface; imaging modality; improved; lens; particle; physical chemical interaction; public health relevance; reconstruction; rice dwarf virus; routine practice; small molecule; software development; structural biology; success; three dimensional structure; tool; usability; virology; virus core

DESCRIPTION (provided by applicant): The long-term objective of this application is to develop methods for the efficient determination of atomic- resolution three-dimensional (3D) structures of large biological complexes in their native, non-crystalline states. The emerging technology of cryo-electron microscopy and 3D reconstruction (collectively "cryoEM") offers great promise for such structural studies. In the current funding period, the PI's group has developed data processing methods, implemented software, and validated these advances by determination of cryoEM structures of a number of large complexes to near-atomic resolution. We hypothesize that improvements in cryoEM technique, coupled with powerful computational tools, can be developed to create and process terabytes of image data for determination of atomic models of large complexes. Whereas we aimed and succeeded in the current funding period to obtain cryoEM structures at near-atomic resolution, the overall goal of this renewal application is to obtain structures at atomic resolution. In the renewal, we propose to improve both imaging itself and computation to bring resolution to 2.5E, permitting identification of amino acids and ultimately atomic resolution. In the renewal period, we therefore focus on improvement of imaging and methods for image acquisition and correction (Aim #1), making cryoEM structure determination hundreds of times more efficient and affordable (Aim #2), extending our success with icosahedral objects to helical ones (Aim #3), improvement of cryoEM-specific atomic-model refinement software that takes advantage of phase as well as amplitude data and that can handle the vastly greater amounts of data required for atomic resolution 3D reconstruction (Aim #4), and validation by applying these new methods for atomic structure determination to a small icosahedral hepatitis B virus (HBV) core, a large icosahedral aquareovirus and the helical tobacco mosaic virus (TMV) (Aim #5). Our overriding principle in achievement of all of these aims is that the optimized methods we create should permit them to be used routinely, reproducibly, and affordably. A successful outcome of this renewal project will remove the final obstacles towards determination of atomic- resolution structures for large complexes by cryoEM and will have great impact on many areas of biomedical research. This achievement will complement other structural methods, particularly X-ray crystallography of purified proteins that are amenable to crystallization and NMR of small molecules in solution. Specifically, this achievement will enable investigators to place individual proteins within the structural context of the larger assembly and to visualize native shapes and physical chemical interactions among the parts of the complex. Moreover, the ability to look at large complexes at atomic resolution will permit visualization of complexes bound to antibodies, receptors, and drugs.

描述（由申请人提供）：本申请的长期目标是开发有效确定其本地非晶状体状态中大生物复合物的原子分辨率三维（3D）结构的方法。冷冻电子显微镜和3D重建的新兴技术（统称为“冷冻”）为此类结构研究提供了巨大的希望。在当前的资金期间，PI的组开发了数据处理方法，实施了软件，并通过确定许多大型复合物的冷冻结构到接近原子分辨率来验证这些进步。我们假设Cryoem技术的改进以及功能强大的计算工具可以创建和处理图像数据的Terabytes，以确定大型复合物的原子模型。尽管我们在当前的融资期间以近原子分辨率获得冷冻结构，但我们以近原子的分辨率获得了成功，但该更新应用的总体目标是以原子分辨率获得结构。在续订中，我们建议将自己的成像和计算提高到2.5E，允许鉴定氨基酸并最终原子分辨率。 In the renewal period, we therefore focus on improvement of imaging and methods for image acquisition and correction (Aim #1), making cryoEM structure determination hundreds of times more efficient and affordable (Aim #2), extending our success with icosahedral objects to helical ones (Aim #3), improvement of cryoEM-specific atomic-model refinement software that takes advantage of phase as well as amplitude data and that can handle the vastly greater原子分辨率3D重建所需的数据量（AIM＃4），以及通过将这些新方法应用于原子结构确定的小型二十面体丙型肝炎病毒（HBV）核心，大型二十面体海虫病毒和螺旋式烟草摩西病毒（TMV）（TMV）（AIM＃5）来验证。我们实现所有这些目标的压倒性原则是，我们创建的优化方法应允许它们常规，可重复和负担得起。该更新项目的成功结果将消除针对Cryoem确定大型复合物的原子分辨率结构的最终障碍，并将对许多生物医学研究的许多领域产生重大影响。这项成就将补充其他结构方法，尤其是纯化蛋白质的X射线晶体学，这些蛋白质可与溶液中小分子的结晶和NMR相提并论。具体而言，这项成就将使研究人员能够将单个蛋白质放置在较大的组装的结构环境中，并可视化综合体部分之间的天然形状和物理化学相互作用。此外，对原子分辨率进行大型复合物的能力将允许可视化与抗体，受体和药物结合的复合物。