INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists of cytoplasmic filaments, a cytoplasmic ring (CR), an inner ring, a nuclear ring, a nuclear basket, and a luminal ring. Each NPC has eight repeating subunits. Structure determination of NPC is a prerequisite for understanding its functional mechanism. In the past two decades, integrative modeling, which combines x-ray structures of individual nucleoporins and subcomplexes with cryo–electron tomography reconstructions, has played a crucial role in advancing our knowledge about the NPC. The CR has been a major focus of structural investigation. The CR subunit of human NPC was reconstructed by cryo–electron tomography through subtomogram averaging to an overall resolution of ~20 Å, with local resolution up to ~15 Å. Each CR subunit comprises two Y-shaped multicomponent complexes known as the inner and outer Y complexes. Eight inner and eight outer Y complexes assemble in a head-to-tail fashion to form the proximal and distal rings, respectively, constituting the CR scaffold. To achieve higher resolution of the CR, we used single-particle cryo–electron microscopy (cryo-EM) to image the intact NPC from the NE of Xenopus laevis oocytes. Reconstructions of the core region and the Nup358 region of the X. laevis CR subunit had been achieved at average resolutions of 5 to 8 Å, allowing identification of secondary structural elements. RATIONALE Packing interactions among the components of the CR subunit were poorly defined by all previous EM maps. Additional components of the CR subunit are strongly suggested by the EM maps of 5- to 8-Å resolution but remain to be identified. Addressing these issues requires improved resolution of the cryo-EM reconstruction. Therefore, we may need to enhance sample preparation, optimize image acquisition, and develop an effective data-processing strategy. RESULTS To reduce conformational heterogeneity of the sample, we spread the opened NE onto the grids with minimal force and used the chemical cross-linker glutaraldehyde to stabilize the NPC. To alleviate orientation bias of the NPC, we tilted sample grids and imaged the sample with higher electron dose at higher angles. We improved the image-processing protocol. With these efforts, the average resolutions for the core and the Nup358 regions have been improved to 3.7 and 4.7 Å, respectively. The highest local resolution of the core region reaches 3.3 Å. In addition, a cryo-EM structure of the N-terminal α-helical domain of Nup358 has been resolved at 3.0-Å resolution. These EM maps allow the identification of five copies of Nup358, two copies of Nup93, two copies of Nup205, and two copies of Y complexes in each CR subunit. Relying on the EM maps and facilitated by AlphaFold prediction, we have generated a final model for the CR of the X. laevis NPC. Our model of the CR subunit includes 19,037 amino acids in 30 nucleoporins. A previously unknown C-terminal fragment of Nup160 was found to constitute a key part of the vertex, in which the short arm, long arm, and stem of the Y complex meet. The Nup160 C-terminal fragment directly binds the β-propeller proteins Seh1 and Sec13. Two Nup205 molecules, which do not contact each other, bind the inner and outer Y complexes through distinct interfaces. Conformational elasticity of the two Nup205 molecules may underlie their versatility in binding to different nucleoporins in the proximal and distal CR rings. Two Nup93 molecules, each comprising an N-terminal extended helix and an ACE1 domain, bridge the Y complexes and Nup205. Nup93 and Nup205 together play a critical role in mediating the contacts between neighboring CR subunits. Five Nup358 molecules, each in the shape of a shrimp tail and named “the clamp,” hold the stems of both Y complexes. The innate conformational elasticity allows each Nup358 clamp to adapt to a distinct local environment for optimal interactions with neighboring nucleoporins. In each CR subunit, the α-helical nucleoporins appear to provide the conformational elasticity; the 12 β-propellers may strengthen the scaffold. CONCLUSION Our EM map–based model of the X. laevis CR subunit substantially expands the molecular mass over the reported composite models of vertebrate CR subunit. In addition to the Y complexes, five Nup358, two Nup205, and two Nup93 molecules constitute the key components of the CR. The improved EM maps reveal insights into the interfaces among the nucleoporins of the CR. Cryo-EM structure of the double-layered CR of the X. laevis NPC. The X. laevis CR, containing eight repeating subunits, is modeled on the basis of cryo-EM reconstructions (top left panel). One CR subunit is shown in two different views to highlight nucleoporins of key interest (bottom left and right panels). The inner and outer Y complexes are colored dark and light gray, respectively. Two Nup205, two Nup93, and five Nup358 molecules are colored blue, red, and purple, respectively.

中文翻译：

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非洲爪蟾核孔复合体细胞质环的结构

引言 核孔复合物 (NPC) 位于核膜 (NE) 上并介导核质货物运输。作为最大的细胞机器之一，脊椎动物 NPC 由细胞质丝、细胞质环 (CR)、内环、核环、核篮和腔环组成。每个 NPC 有八个重复的亚基。NPC的结构确定是了解其功能机制的先决条件。在过去的二十年中，将单个核孔蛋白和亚复合物的 X 射线结构与冷冻电子断层扫描重建相结合的综合建模在增进我们对 NPC 的了解方面发挥了至关重要的作用。CR一直是结构调查的主要焦点。人 NPC 的 CR 亚基通过低温电子断层扫描重建，通过亚断层图像平均到 ~20 Å 的整体分辨率，局部分辨率高达 ~15 Å。每个 CR 亚基包含两个 Y 形多组分复合物，称为内部和外部 Y 复合物。八个内部和八个外部 Y 复合物以头对尾的方式组装，分别形成近端和远端环，构成 CR 支架。为了获得更高的 CR 分辨率，我们使用单粒子低温电子显微镜 (cryo-EM) 对来自 NE 的完整 NPC 进行成像 分别构成CR支架。为了获得更高的 CR 分辨率，我们使用单粒子低温电子显微镜 (cryo-EM) 对来自 NE 的完整 NPC 进行成像 分别构成CR支架。为了获得更高的 CR 分辨率，我们使用单粒子低温电子显微镜 (cryo-EM) 对来自 NE 的完整 NPC 进行成像非洲爪蟾卵母细胞。核心区域和 Nup358 区域的重建X. laevisCR 亚基的平均分辨率为 5 到 8 Å，可以识别二级结构元素。基本原理 CR 亚基成分之间的包装相互作用在以前的所有 EM 图中都没有很好地定义。5 到 8 Å 分辨率的 EM 图强烈建议 CR 亚基的其他成分，但仍有待确定。解决这些问题需要改进冷冻电镜重建的分辨率。因此，我们可能需要加强样品制备、优化图像采集并制定有效的数据处理策略。结果 为了减少样品的构象异质性，我们以最小的力将打开的 NE 散布到网格上，并使用化学交联剂戊二醛来稳定 NPC。为了减轻NPC的方向偏差，我们倾斜样品网格并以更高的角度对具有更高电子剂量的样品进行成像。我们改进了图像处理协议。通过这些努力，核心和 Nup358 区域的平均分辨率已分别提高到 3.7 和 4.7 Å。核心区域的最高局部分辨率达到 3.3 Å。此外，Nup358 的 N 端 α-螺旋结构域的冷冻电镜结构已在 3.0-Å 分辨率下得到解析。这些 EM 图允许识别 Nup358 的五个副本、两个 Nup93 副本、两个 Nup205 副本以及每个 CR 亚基中的两个 Y 复合物副本。依靠 EM 地图并在 AlphaFold 预测的帮助下，我们生成了最终的 CR 模型 核心和 Nup358 区域的平均分辨率分别提高到 3.7 和 4.7 Å。核心区域的最高局部分辨率达到 3.3 Å。此外，Nup358 的 N 端 α-螺旋结构域的冷冻电镜结构已在 3.0-Å 分辨率下得到解析。这些 EM 图允许识别 Nup358 的五个副本、两个 Nup93 副本、两个 Nup205 副本以及每个 CR 亚基中的两个 Y 复合物副本。依靠 EM 地图并在 AlphaFold 预测的帮助下，我们生成了最终的 CR 模型 核心和 Nup358 区域的平均分辨率分别提高到 3.7 和 4.7 Å。核心区域的最高局部分辨率达到 3.3 Å。此外，Nup358 的 N 端 α-螺旋结构域的冷冻电镜结构已在 3.0-Å 分辨率下得到解析。这些 EM 图允许识别 Nup358 的五个副本、两个 Nup93 副本、两个 Nup205 副本以及每个 CR 亚基中的两个 Y 复合物副本。依靠 EM 地图并在 AlphaFold 预测的帮助下，我们生成了最终的 CR 模型 这些 EM 图允许识别 Nup358 的五个副本、两个 Nup93 副本、两个 Nup205 副本以及每个 CR 亚基中的两个 Y 复合物副本。依靠 EM 地图并在 AlphaFold 预测的帮助下，我们生成了最终的 CR 模型 这些 EM 图允许识别 Nup358 的五个副本、两个 Nup93 副本、两个 Nup205 副本以及每个 CR 亚基中的两个 Y 复合物副本。依靠 EM 地图并在 AlphaFold 预测的帮助下，我们生成了最终的 CR 模型X. laevis全国人大。我们的 CR 亚基模型包括 30 个核孔蛋白中的 19,037 个氨基酸。一个以前未知的 Nup160 C 末端片段被发现构成了顶点的关键部分，其中 Y 复合体的短臂、长臂和茎相遇。Nup160 C 末端片段直接结合 β-螺旋桨蛋白 Seh1 和 Sec13。两个不相互接触的 Nup205 分子通过不同的界面结合内部和外部 Y 复合物。两个 Nup205 分子的构象弹性可能是它们与近端和远端 CR 环中不同核孔蛋白结合的多功能性的基础。两个 Nup93 分子，每一个都包含一个 N 端延伸的螺旋和一个 ACE1 结构域，桥接 Y 复合物和 Nup205。Nup93 和 Nup205 在调解相邻 CR 亚基之间的接触方面发挥着关键作用。五个 Nup358 分子，每个都呈虾尾的形状，被命名为“夹子”，握住两个 Y 复合物的茎。与生俱来的构象弹性允许每个 Nup358 夹适应不同的局部环境，以实现与相邻核孔蛋白的最佳相互作用。在每个 CR 亚基中，α-螺旋核孔蛋白似乎提供了构象弹性。12个β-螺旋桨可以加强脚手架。结论 我们基于 EM 地图的模型 12个β-螺旋桨可以加强脚手架。结论 我们基于 EM 地图的模型 12个β-螺旋桨可以加强脚手架。结论 我们基于 EM 地图的模型X. laevisCR 亚基大大扩展了脊椎动物 CR 亚基复合模型的分子量。除了 Y 复合物外，五个 Nup358、两个 Nup205 和两个 Nup93 分子构成了 CR 的关键成分。改进的 EM 图揭示了对 CR 核孔蛋白之间界面的深入了解。 双层 CR 的冷冻电镜结构X. laevis全国人大。这X. laevisCR 包含八个重复的亚基，基于冷冻电镜重建（左上图）建模。一个 CR 亚基显示在两个不同的视图中，以突出显示关键感兴趣的核孔蛋白（左下和右下图）。内部和外部 Y 配合物分别着色为深灰色和浅灰色。两个 Nup205、两个 Nup93 和五个 Nup358 分子分别为蓝色、红色和紫色。