High-Resolution CryoEM Reconstruction of Large Complexes

大型复合物的高分辨率冷冻电镜重建

基本信息

批准号：
10813503
负责人：
Z Hong ZHOU
金额：
$ 8.53万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
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/10813503
关键词：
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 Cytoskeletal Filaments DNA DNA Viruses Data Development Disease Double Stranded DNA Virus Double Stranded RNA Virus Drug Design Electrons Erythrocytes Flagella Funding Genetic Transcription Genome Geometry Goals Heart Human Image In Situ Individual Investigation Learning Lipids Macromolecular Complexes Maintenance Maps Mass Spectrum Analysis Membrane Proteins Methods Modeling Molecular Nucleic Acids Outcome Polymers 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 Visualization X-Ray Crystallography bacteriocin biological systems cell motility computerized data processing convolutional neural network deep learning density design genetic information nanomachine neural network new therapeutic target non-Native novel nucleic acid structure particle polymerization programs protein complex protein purification protein transport quantum rational design reconstruction routine practice small molecule structural biology success three dimensional structure tool transmission process

项目摘要

PROJECT SUMMARY 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.

项目概要总体而言，该项目旨在开发方法，以原子分辨率有效确定三维大型、天然、对称不匹配、局部无序或多态生物复合体的 (3D) 结构通过冷冻电子显微镜（cryoEM），该项目过去的资助周期（2014-2019）非常引人注目。结构生物学领域的巨大变革，通常被称为“冷冻电镜革命”。单粒子冷冻电镜可以说已成为结构生物学的默认选择。该项目使 PI 团队能够在多个方面为这一转变做出贡献：我们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大型二十面体和螺旋复合体中重要的（有时是基本的）生物过程，如以及纯化的蛋白质-核酸复合物和膜蛋白的原子模型我们一直在努力将电子计数结合起来。冷冻电镜（即量子）功能可以通过确定天然细胞的原子模型来推进该领域的发展复合物、病毒基因组包装和原位转录以及瞬时稳定的催化中间体。在上一个资助期间，我们的目标是使用我们的冷冻电镜导出原子模型，并成功地获得了该模型单独的方法适用于较大的复合物，特别是二十面体和螺旋病毒。现在，这种更新。该申请旨在开发一种用于细胞原子结构的综合蛋白质组冷冻电镜方法我们还将使用病毒作为解决问题的工具。有关基因组包装、压缩、超螺旋、复制和转录的基本问题。我们将继续开发亚颗粒细化和核酸的新型计算方法建模，特别是cryoID，这是一个集成软件包，可实现近原子分辨率的cryoEM 丰富的细胞环境中许多复合物的结构待确定、鉴定和原子化建模（目标#1）；大变形中的原位原子结构的亚粒子重建和细化或本质上结构异质的复杂包膜，例如大型病毒和天然病毒中的包膜细胞复合物（例如螺旋组装体）（目标#2）；对 RNA 和 DNA 中的基因组结构进行建模病毒以及细胞转录/复制复合物（目标#3）；并验证这些新方法应用于红细胞蛋白质组、易位细菌素纳米机器的原子结构测定和膜蛋白复合物、螺旋丝状细胞（例如肌动蛋白和轴丝）和病毒组装体以及许多 ssRNA、dsRNA 和 dsDNA 病毒内部的基因组结构（目标#4）。该更新项目的成功将进一步推动冷冻电镜在大型结构研究中的应用。这一成果将推动生物医学研究的许多领域的发展。冷冻电镜的当前限制并补充其他结构方法，特别是 X 射线晶体学纯化蛋白质和溶液中小分子的 NMR 高度纯化分子的方法。