Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK.
Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
EMBO J. 2021 Apr 15;40(8):e106164. doi: 10.15252/embj.2020106164. Epub 2021 Mar 18.
Dynactin is a 1.1 MDa complex that activates the molecular motor dynein for ultra-processive transport along microtubules. In order to do this, it forms a tripartite complex with dynein and a coiled-coil adaptor. Dynactin consists of an actin-related filament whose length is defined by its flexible shoulder domain. Despite previous cryo-EM structures, the molecular architecture of the shoulder and pointed end of the filament is still poorly understood due to the lack of high-resolution information in these regions. Here we combine multiple cryo-EM datasets and define precise masking strategies for particle signal subtraction and 3D classification. This overcomes domain flexibility and results in high-resolution maps into which we can build the shoulder and pointed end. The unique architecture of the shoulder securely houses the p150 subunit and positions the four identical p50 subunits in different conformations to bind dynactin's filament. The pointed end map allows us to build the first structure of p62 and reveals the molecular basis for cargo adaptor binding to different sites at the pointed end.
动力蛋白激活因子是一个 1.1 MDa 的复合物，可激活分子马达动力蛋白，以实现沿微管的超长距离运输。为了实现这一目标，它与动力蛋白和卷曲螺旋接头形成了一个三元复合物。动力蛋白激活因子由一个肌动蛋白相关的细丝组成，其长度由其灵活的肩部结构域定义。尽管有先前的冷冻电镜结构，但由于这些区域缺乏高分辨率信息，因此仍然不清楚细丝的肩部和尖端的分子结构。在这里，我们结合了多个冷冻电镜数据集，并定义了精确的掩蔽策略，用于粒子信号的减除和 3D 分类。这克服了结构域的灵活性，并产生了高分辨率的图谱，我们可以在其中构建肩部和尖端。肩部的独特结构安全地容纳了 p150 亚基，并将四个相同的 p50 亚基置于不同的构象中，以结合动力蛋白激活因子的细丝。尖端的图谱使我们能够构建 p62 的第一个结构，并揭示了货物接头与尖端不同部位结合的分子基础。
