Structure and function of Arp 2/3 complex--Subproject 3

Arp2/3复合物在运动细胞的前沿以位点定向、信号控制的方式形成新丝。这导致了密集的分支肌动蛋白网络的形成，这种网络被认为提供了驱动板足突出的力量。本文的研究旨在确定Arp2/3复合物在激活和分支结形成过程中的三维结构和结构变化。电子冷冻显微镜和各种图像分析方法的结合将用于提供arp2 /3复合物在其自由，活化和肌动蛋白结合形式的三维重建。这将辅以亚基定位使用金标。由于f -肌动蛋白的丝状性质排除了传统的晶体学方法，上述方法提供了单个蛋白质的结构信息与其在不同结构复杂性水平的肌动蛋白结合形式中的结构之间的唯一联系。晶体结构与电子显微镜重建的对接将允许将单个组件的原子分辨率信息与它们的多复杂形式相关联。

The Arp2/3 complex nucleates new filaments in site-directed, signal-controlled fashion at the leading edge of motile cells. This gives rise to the formation of dense, branched actin networks that are thought to provide the force that drives lamellipodia protrusion. The studies proposed here aim at determining the three-dimensional structure and structural changes of the Arp2/3 complex occurring during activation and branch junction formation. A combination of electron cryomicroscopy and various image analysis approaches will be used to provide three-dimensional reconstructions of theArp2/3 complex in its free, activated and actin-bound forms. This will be complemented by subunit localization using gold labeling. As the filamentous nature of F-actin precludes conventional crystallographic methods, the above approaches provide the only link between structural information of the individual proteins and their structure in the actin-bound forms at various levels of structural complexity. Docking of the crystal structures into the electron microscopic reconstructions will allow to correlate atomic resolution information of the individual components to their multi-complex form.