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: 10367917
• 负责人: Z Hong ZHOU
• 金额: $ 32.09万
• 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 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10367917
• 关键词: 3-Dimensional; Achievement; Actins; Address; Amino Acids; Area; Binding; Biological; Biological Process; Biomedical Research; Cell physiology; Cells; Cellular Structures; Chemicals; Classification; Code; Complement; Complex; Computer Models; Computer software; Computing Methodologies; Cryoelectron Microscopy; Cytoskeleton; DNA; DNA Viruses; Data; Development; Double Stranded DNA Virus; Double Stranded RNA Virus; Drug Design; Electrons; Erythrocytes; Filament; Flagella; Funding; Genetic Transcription; Genome; Geometry; Goals; Heart; Human; In Situ; Individual; Investigation; Learning; Lipids; Macromolecular Complexes; Maintenance; Maps; Mass Spectrum Analysis; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Nucleic Acids; Outcome; Process; Proteins; Proteome; Proteomics; RNA; RNA Viruses; Research Personnel; Resistance; Resolution; Rod; Scheme; Source; Structure; Techniques; Testing; Therapeutic Intervention; Training; Transcription Process; Vaccines; Validation; Viral; Viral Genome; Virus; Virus Assembly; Virus Replication; X-Ray Crystallography; bacteriocin; biological systems; cell motility; computerized data processing; convolutional neural network; deep learning; density; design; genetic information; image processing; nanomachine; neural network; new therapeutic target; non-Native; novel; nucleic acid structure; particle; polymerization; programs; protein complex; protein transport; quantum; rational design; reconstruction; routine practice; small molecule; structural biology; success; three dimensional structure; tool; transmission process; virus envelope

Project Summary/Abstract Overall, this project aims to develop methods to efficiently determine, at atomic-resolution, three-dimensional (3D) structures of large, native, symmetry-mismatched, locally dis-ordered or polymorphic biological complexes through cryo electron microscopy (cryoEM). The past funding cycle of this project (2014-2019) has witnessed a drastic transformation of the field of structural biology, popularly referred as “the cryoEM revolution”. Today, single-particle cryoEM has arguably become the default choice for structural biology. The funding from this project has enabled the PI’s group to contribute to this transformation in multiple fronts: we have developed novel imaging and processing methods and validated these methods by determining in situ structures of important—and sometimes fundamental—biological processes in large icosahedral and helical complexes, as well as atomic models of purified protein-nucleic acid complexes and membrane proteins that have been resistant to previous x-ray crystallography and NMR efforts. We hypothesize that combining electron counting (i.e., quantum) capabilities with cryoEM can advance the field by determining atomic models of native cellular complexes, viral genome packaging and transcription in situ and transiently stable catalytic intermediates. In the last funding period we aimed to, and succeeded in, derive atomic models using our cryoEM method alone for larger complexes, particularly those in icosahedral and helical viruses. Now, this renewal application aims to develop an integrative proteome cryoEM method for atomic structures of cellular complexes in their native, unpurified, and functional states. We will also use viruses as tools to address fundamental questions concerning genome packaging, compaction, supercoiling, replication and transcription. We will continue developing novel computational methods for sub-particle refinement and nucleic acid modeling, specifically cryoID, an integrative software package that allows near-atomic resolution cryoEM structures from many complexes in enriched cellular milieu to be determined, identified, and atomically modeled (Aim #1); sub-particle reconstructions and refinement for in situ atomic structures in large deformable or intrinsically structurally heterogeneous complexes such as those in large enveloped viruses and native cellular complexes (e.g., helical assemblies) (Aim #2); modeling genome structures in both RNA and DNA viruses, as well as cellular transcriptional/replicative complexes (Aim #3); and validate these new methods for atomic structure determination by application to red blood cell proteome, translocon bacteriocin nano-machines and membrane protein complexes, helical filamentous cellular (e.g., actin and axoneme) and viral assemblies and genome structures inside a number of ssRNA, dsRNA and dsDNA viruses (Aim #4). A successful outcome of this renewal project will further advance cryoEM in structural studies of large complexes and will have great impact on many areas of biomedical research. This achievement will push the current limits of cryoEM and complement other structural methods, particularlythe X-ray crystallography of purified proteins and NMR of small molecules in solutionmethods of highly purified molecules.

项目摘要/摘要 总体而言，该项目旨在开发方法以有效地确定原子分辨率的三维方法 （3D）大型，天然，对称性不匹配的，局部分歧或多态生物学复合物的结构 通过冷冻电子显微镜（冷冻）。该项目的过去筹资周期（2014- 2019年）见证了 结构生物学领域的急剧转变，通常称为“冷冻革命”。今天， 单粒子冷冻可以说是结构生物学的默认选择。从中的资金 项目使PI小组能够在多个方面为这种转变做出贡献：我们已经开发了 新颖的成像和处理方法，并通过确定原位结构来验证这些方法 重要的，有时甚至是基本的 - 大型二十面体和螺旋络合物中的生物学过程，如 以及纯化蛋白核酸复合物和膜蛋白的原子模型 抵抗以前的X射线晶体学和NMR努力。我们假设将电子计数结合在一起 （即，量子）与冷冻的能力可以通过确定天然细胞的原子模型来推进田地 复合物，病毒基因组包装和原位转录以及瞬时稳定的催化中间体。 在最后的融资期内，我们旨在使用我们的冷冻物来得出原子模型 仅用于较大复合物，尤其是二十面体和螺旋病毒的方法。现在，这个更新 应用旨在开发用于细胞原子结构的集成蛋白质组冷冻法 其本地，未繁殖和功能状态的复合物。我们还将使用病毒作为解决的工具 有关基因组包装，压实，超螺旋，复制和转录的基本问题。 我们将继续开发用于细分和核酸的新型计算方法 建模，特别是Cryoid，是一种允许接近原子分辨率冷冻的集成软件包 来自富集的细胞环境中许多复合物的结构，以确定，鉴定和原子 建模（AIM＃1）;较大变形的原位原子结构的亚颗粒重建和改进 或本质上结构上异质的复合物，例如大型包围病毒和天然的复合物 细胞复合物（例如，螺旋组件）（AIM＃2）;在RNA和DNA中对基因组结构进行建模 病毒以及细胞转录/复制复合物（AIM＃3）；并验证这些新方法的 通过应用于红细胞蛋白质组，易位细菌纳米机器的原子结构测定 和膜蛋白复合物，螺旋丝状细胞（例如肌动蛋白和轴突）和病毒组件 以及许多ssRNA，dsRNA和dsDNA病毒中的基因组结构（AIM＃4）。 这个更新项目的成功结果将进一步推进大型结构研究的冷冻 复合物，将对生物医学研究的许多领域产生重大影响。这项成就将推动 当前的冷冻限制并补充其他结构方法，尤其是X射线晶体学 高纯化分子的溶液方法中的小分子的纯化蛋白质和NMR。