Project #1: Harrison

项目

• 批准号: 8462406
• 负责人: STEPHEN COPLAN HARRISON
• 金额: $ 75.9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462406
• 关键词: Architecture; Binding; Biological; C-Microtubule; Capsid; Cell division; Cells; Chromosome Segregation; Chromosomes; Clathrin; Collaborations; Complex; Computing Methodologies; Cryoelectron Microscopy; Crystallography; Cytosol; DNA; Electron Microscopy; Event; Goals; Image; Imaging technology; In Vitro; Individual; Instruction; Intracellular Membranes; Investigation; Kinetochores; Life; Link; Maps; Mediating; Membrane; Methods; Microtubules; Mitotic spindle; Modification; Molecular; Molecular Machines; Molecular Models; Pathway interactions; Penetration; Preparation; Procedures; Process; Proteins; Recombinant Proteins; Recombinants; Recruitment Activity; Resolution; Rotavirus; Saccharomycetales; Staining method; Stains; Structure; Surface; System; Testing; Toxin; Vesicle; Viral; Virion; Virus; Work; Yeasts; base; cellular imaging; conformational conversion; density; fluorescence imaging; image reconstruction; insight; member; molecular modeling; particle; pathogen; programs; reconstitution; reconstruction; research study; uptake

.PROJECT SUMMARY (See instructions): This component of the Program Project is a proposal to study structure and assembly of three intricate molecular machines ~ a non-enveloped virus, clathrin coats, and kinetochores. (1) A long-term goal of our work on non-enveloped viruses and on clathrin coats has been to connect high-resolution structure analysis (by x-ray crystallography and by single-particle cryoEM) with observations on the dynamics of individual particles by fluorescence imaging. CryoEM structures of rotavirus particles at near-atomic resolution and of clathrin coats at subnanometer resolution, determined in collaboration with members of the Program Project team, have led to experiments that define mechanisms of viral entry and clathrin assembly and uncoating, both in vitro and in living cells. We will now use cryoET to bridge between high-resolution structural analyses and live-cell imaging, in collaboration with Project 4. The objectives are to determine how a non-enveloped virus (rotavirus in the work here) perforates or disrupts the membrane of an intracellular vesicle in order to gain access to the cytosol ~ a critical and still puzzling step in the infectious process ~ and to visualize interactions of clathrin with other coat components. (2) Budding-yeast kinetochores assemble on centromeric DNA (-150 bp) and connect each chromosome to a single microtubule (MT) of the mitotic spindle. We have reconstituted the DNA-proximal "inner kinetochore" from recombinant proteins in vitro, and we have determined crystal structures of many of its components. We will obtain 3D reconstructions, from negatively stained preparations and by cryoEM where appropriate, of the reconstituted inner kinetochore and some of its subcomplexes, and build a molecular model of this -750 kDa protein:DNA assembly. From analysis of the inner-kinetochore structure and information on its interaction partners, we will add additional "linker" complexes, which connect the inner kinetochore with microtubule-attached "outer kinetochore" components, and determine further structures by EM

.项目摘要（参见说明）：该计划项目的这一部分是研究三种复杂分子机器（无包膜病毒、网格蛋白外壳和着丝粒）的结构和组装的提案。 (1) 我们在无包膜病毒和网格蛋白外壳方面的工作的长期目标是将高分辨率结构分析（通过 X 射线晶体学和单粒子冷冻电镜）与通过荧光成像对单个粒子的动力学观察联系起来。与该计划项目团队成员合作确定了近原子分辨率的轮状病毒颗粒的冷冻电镜结构和亚纳米分辨率的网格蛋白涂层的冷冻电镜结构，这导致了在体外和活细胞中定义病毒进入以及网格蛋白组装和脱壳机制的实验。现在，我们将与项目 4 合作，使用 CryoET 在高分辨率结构分析和活细胞成像之间架起桥梁。目标是确定无包膜病毒（此处工作中的轮状病毒）如何穿孔或破坏细胞内囊泡的膜，以便进入胞质溶胶（感染过程中的一个关键且仍然令人费解的步骤），并可视化网格蛋白与其他蛋白的相互作用 涂层组件。 (2) 出芽酵母着丝粒在着丝粒 DNA (-150 bp) 上组装，并将每条染色体连接到有丝分裂纺锤体的单个微管 (MT)。我们已经在体外从重组蛋白重建了DNA近端“内着丝粒”，并且我们已经确定了其许多成分的晶体结构。我们将从负染色制剂中并在适当的情况下通过冷冻电镜获得重建的内部动粒及其一些子复合体的 3D 重建，并构建此 -750 kDa 蛋白质：DNA 组装的分子模型。通过分析内动粒结构及其相互作用伙伴的信息，我们将添加额外的“连接器”复合物，将内动粒与微管附着的“外动粒”组件连接起来，并通过 EM 确定进一步的结构