Structure

结构

• 批准号: 8121501
• 负责人: ROBERT Colin LIDDINGTON
• 金额: $ 49.57万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8121501
• 关键词: Actinin; Actins; Adhesions; Binding; Binding Sites; Cells; Complex; Data; Docking; Electron Microscopy; Goals; Image; Immigration; Individual; Integrins; Life; Light; Maps; Mediating; Methods; Microscopy; Organelles; Phase; Proteins; Resolution; Roentgen Rays; Structure; Three-dimensional analysis; Vinculin; base; light microscopy; migration; three dimensional structure; tomography

The continued goal of this Initiative is to determine the high resolution structures of two major migration organelles, adhesions and protrusions. Our approach is to combine structural methods that range from correlative light and high resolution electron microscopy to X-ray and NMR. As part of this effort, we are developing a new imaging concept that allows us to capture by light microscopy the spatial-temporal aspects of migration in living cells and to visualize the same structures using high resolution electron microscopy - 3D correlative light and electron microscopy ("top down approaches"). High resolution structures of individual proteins and their detailed interactions with binding partners obtained by NMR, X-ray or tomography ("bottom up approaches") can then be computationally docked into the three-dimensional maps of the complexes. In the first phase of the Consortium, this paradigm was applied to the structure of the actin network in protrusions, providing 3D structures of Arp2/3 mediated actin branches and the structural basis for Arp2/3 activation. We also determined the ultrastructure of the lamellipodia of living cells using our new 3D correlative EM and speckle microscopy and determined the vinculin binding site on a-actinin in the presence of integrin. A unified framework for segmentation and analysis of 3-D data was developed to enable the identification of assemblies in 3-D structures.

该计划的持续目标是确定两个主要的高分辨率结构 迁移细胞器，粘附和突起。我们的方法是结合结构方法 从相关光和高分辨率电子显微镜到X射线和NMR。作为这项努力的一部分，我们是 开发一个新的成像概念，使我们能够通过光学显微镜捕获 在活细胞中迁移并使用高分辨率电子显微镜可视化相同的结构-3D 相关光和电子显微镜（“自上向下接近”）。个人的高分辨率结构 蛋白质及其与NMR，X射线或层析成像获得的结合伴侣的详细相互作用（底部 然后可以将其计算到复合物的三维图中。 财团的第一阶段，将此范式应用于肌动蛋白网络在突起中的结构， 提供ARP2/3介导的肌动蛋白分支的3D结构和ARP2/3激活的结构基础。我们 还使用我们的新3D相关EM和 在整合素的存在下，斑点显微镜并确定了a-肌动蛋白上的小蛋白结合位点。一个统一 开发了用于分割和分析3-D数据的框架，以实现组件的识别 在3-D结构中。