Structure

结构

• 批准号: 7195624
• 负责人: ROBERT Colin LIDDINGTON
• 金额: $ 29.51万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7195624
• 关键词: X ray crystallography; actins; biomedical facility; calorimetry; cell adhesion; cell adhesion molecules; cell migration; chemical kinetics; computer simulation; cooperative study; cryoscopy; crystallization; cytoskeletal proteins; electron microscopy; fluorescence microscopy; intermolecular interaction; model design /development; molecular assembly /self assembly; nuclear magnetic resonance spectroscopy; physical model; polymerization; protein structure function; structural biology; thermodynamics; time resolved data; 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.

该倡议的持续目标是确定两个主要的高分辨率结构 迁移细胞器、粘连和突起。我们的方法是结合各种结构方法 从关联光和高分辨率电子显微镜到 X 射线和核磁共振。作为这项努力的一部分，我们正在 开发一种新的成像概念，使我们能够通过光学显微镜捕获物体的时空方面 活细胞中的迁移并使用高分辨率电子显微镜 - 3D 观察相同的结构 相关光学和电子显微镜（“自上而下的方法”）。个体的高分辨率结构 通过 NMR、X 射线或断层扫描获得的蛋白质及其与结合伴侣的详细相互作用（“底部 向上的方法”）然后可以通过计算对接到复合物的三维图中。 在联盟的第一阶段，该范式被应用于突起中肌动蛋白网络的结构， 提供 Arp2/3 介导的肌动蛋白分支的 3D 结构以及 Arp2/3 激活的结构基础。我们 还使用我们新的 3D 相关 EM 确定了活细胞片状伪足的超微结构 散斑显微镜并确定了在整联蛋白存在的情况下α-辅肌动蛋白上的纽蛋白结合位点。统一的 开发了 3D 数据分割和分析框架，以便能够识别组件 在 3-D 结构中。