Supplement: Atomic Structure of the Nuclear Pore Complex

这项设备补充并不改变资助研究的范围，而是取代了至关重要的 及时实现原定目标的设备。因此，原始摘要是 包括在内。 摘要 遗传信息被包围在细胞核中是进化的最大标志之一，但 需要专门的门户，折叠的蛋白质和蛋白质/核酸 复合体可以穿过核膜(NE)。核孔复合体(NPC)，一种圆柱形 嵌入在贯穿NE的圆形孔中的超分子结构是 通过NE，并可完成大分子的选择性双向传输 直径可达~40纳米，速度为每秒数百个事件。除了它的主要角色之外 核质运输，鼻咽癌还有助于额外的基因调控模式 例如，通过与转录和信使核糖核酸输出机制直接相互作用。全国人大就是这样 代表着所有真核生物生命所必需的细胞器，因此，鼻咽癌功能障碍 与各种形式的人类疾病有关。在建筑上，全国人大由一个中央 被称为细胞质细丝和核篮的不对称成分的对称核心 附在这里。NPC是由大约34种不同的称为核孔素的蛋白质组成的，每种蛋白质都存在于 多个拷贝，使整个组装达到人类体内~110个丙二醛的非凡质量。 核孔蛋白被组织成不同的亚复合体，这些亚复合体构成了 活体完整的鼻咽癌。为了确定全国人大的原子结构，我的小组一直在研究一种 分而治之的方法，其中我们已经绘制了核孔素相互作用图，重新构建 重组核孔蛋白络合物及其低温电子结构的确定 完整鼻咽癌的断层重建。通过这种方式，我们实现了一种近原子的复合材料 上一次授权期内~60MDa人NPC对称核的结构。以此为基础 为了取得进展，我们现在建议将我们的结构定性扩大到仍未解决的全国人大部分 并利用我们已经获得的知识来研究与鼻咽癌相关的基本细胞生物学 问题。具体地说，我们计划阐明NPC内环中的分子相互作用 对于其中央传输通道的形成以及对称核心和 跨膜鼻咽癌成分，对于鼻咽癌在东北孔洞中的锚定是必不可少的。结果是 这项拟议的研究有望大大增加我们对分子的了解 鼻咽癌调节核质转运及相关细胞的机制 过程，同时为目前无法治疗的“nup疾病”创造一个机制基础。 此外，这里开发的方法论将作为表征 其他像NPC一样大、灵活和复杂的基本细胞巨型组件，其功能是 由于缺乏结构性洞察力，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.