Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)^1,2,3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel^4,5,6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that—compared with previous human NPC models obtained from purified NEs—the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

核孔复合体 (NPC) 为跨越核包膜 (NE) ^1,2,3的细胞核和细胞质之间的货物运输创造了大型管道。这些多兆道尔顿结构由大约三十种不同的核孔蛋白组成，这些核孔蛋白分布在中央运输通道周围的三个主要亚结构（内环、细胞质环和核质环）中^4,5,6. 在这里，我们在 DLD-1 细胞上使用冷冻电子断层扫描，这些细胞是使用冷冻聚焦离子束铣削制备的，以在其自然环境中生成人类 NPC 的结构模型。我们表明——与之前从纯化的 NEs 获得的人类 NPC 模型相比——我们模型中的内环要宽得多；中央通道体积增加75%，核质环和细胞质环重组。此外，NPC 膜在内环复合体周围表现出不对称性。使用 Nup96 的靶向降解，细胞质和核质环的支架核孔蛋白，我们观察到每个环在调节中央通道和维持膜不对称性方面的相互依赖性。