Supplement: Atomic Structure of the Nuclear Pore Complex

这种设备补充并不改变资助研究的范围，但取代了重要的 设备，及时实现最初的目标。因此，原始摘要是 包括. 摘要 遗传信息被包裹在细胞核中是进化的重要标志之一， 创造了专门的门户的必要性，通过这些门户，折叠的蛋白质和蛋白质/核酸 复合物可以穿过核膜（NE）。核孔复合体（NPC），一个圆柱形的 超分子结构嵌入在渗透NE的圆孔中，是 通过NE，并可实现大分子的选择性双向转运， 直径高达~40 nm，速率为每秒几百个事件。除了其主要作用之外， 除了核质转运外，NPC还有助于其他基因调控模式， 例如，通过与转录和mRNA输出机制的直接相互作用。因此，NPC 代表了所有真核生物的基本细胞器，因此，NPC功能障碍已经被 与各种人类疾病有关。在建筑上，NPC由一个中央 对称核心，称为细胞质丝和核篮的不对称成分 附NPC由约34种不同的蛋白质组成，称为核孔蛋白，每种蛋白质都存在于 多个拷贝，使得整个组装体在人类中达到~110 MDa的非凡质量。 核孔蛋白被组织成不同的亚复合物，其构成核孔蛋白的生理结构单元。 体内完整的NPC。为了确定NPC的原子结构，我的团队一直在研究 分而治之的方法，其中我们已经映射核孔蛋白相互作用，重建 重组核孔蛋白复合物，并确定其晶体结构，以适应低温电子 完整NPC的断层重建。通过这种方式，我们实现了一种近原子复合材料， 在前一个资助期内，~ 60 MDa人类NPC对称核的结构。在此基础上 随着进展，我们现在建议将我们的结构表征扩展到NPC中尚未解决的部分 并利用我们已经获得的知识来解决基本的NPC相关细胞生物学 问题.具体来说，我们计划阐明NPC内环中的分子相互作用， 对于其中央运输通道的形成至关重要，并且在对称核心和 这些跨膜NPC组分对于NPC锚定在NE孔中是必需的。成果 预计这项拟议的研究将大大增加我们对分子的了解 NPC调节核质转运和相关细胞的机制 过程，同时为目前无法治疗的“nup疾病”创造一个机械基础。 此外，本文开发的方法将作为表征 其他重要的细胞巨型组件，如NPC一样大，灵活和复杂，其功能 由于缺乏对结构的了解，机制仍然难以捉摸。

This equipment supplement does not modify the scope of the funded research but replaces vital equipment to realize the original aims in a timely manner. As such, the original abstract is included. Abstract The enclosure of genetic information in the nucleus is one of the great hallmarks of evolution, but creates the necessity for dedicated portals through which folded proteins and protein/nucleic acid complexes can cross the nuclear envelope (NE). The nuclear pore complex (NPC), a cylindrical supramolecular structure embedded in circular pores permeating the NE, is the sole gateway for passage through the NE and can accomplish the selective bidirectional transport of macromolecules of up to ~40 nm in diameter at a rate of several hundred events per second. Beyond its primary role in nucleocytoplasmic transport, the NPC also contributes to additional modes of gene regulation for example through direct interaction with the transcription and mRNA export machineries. The NPC thus represents an essential organelle for all eukaryotic life and, accordingly, NPC dysfunction has been associated with various forms of human disease. Architecturally, the NPC consists of a central symmetric core to which asymmetric components called cytoplasmic filaments and nuclear basket are attached. The NPC is built from ~34 different proteins termed nucleoporins that are each present in multiple copies such that the entire assembly reaches the extraordinary mass of ~110 MDa in humans. Nucleoporins are organized into distinct subcomplexes which constitute physiological building blocks of the intact NPC in vivo. To determine the atomic architecture of the NPC, my group has been pursuing a divide-and-conquer approach, in which we have mapped nucleoporin interactions, reconstituted recombinant nucleoporin complexes and determined their crystal structures to be fit into cryo-electron tomographic reconstructions of the intact NPC. In this way, we achieved a near-atomic composite structure of the ~60MDa human NPC symmetric core in the previous grant period. Building on this progress, we now propose to expand our structural characterization to still unresolved parts of the NPC and to use our already gained knowledge to address fundamental NPC-associated cell biological questions. Specifically, we plan to elucidate the molecular interactions in the NPC’s inner ring that are essential for the formation of its central transport channel, and between the symmetric core and transmembrane NPC components that are essential for NPC anchoring in the NE pores. The outcome of the proposed research is expected to greatly increase our understanding of the molecular mechanisms by which the NPC regulates nucleocytoplasmic transport and associated cellular processes, while simultaneously creating a mechanistic basis for currently untreatable “nup diseases.” Furthermore, the methodologies developed herein will serve as a paradigm for the characterization of other essential cellular mega-assemblies as large, flexible and complex as the NPC whose functional mechanisms have remained elusive due to lack of structural insight.