LAUSR

Description INTRODUCTION The eukaryotic nucleus protects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the largest protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaffold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conformational breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial challenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integrative modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous structural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaffold is interconnected by a set of intrinsically disordered linker NUPs that are not straightforwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topology and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)–based prediction to generate an extensive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharacterized. Benchmarking against previous and unpublished x-ray and cryo–electron microscopy structures revealed unprecedented accuracy. We obtained well-resolved cryo–electron tomographic maps of both the constricted and dilated conformational states of the human NPC. Using integrative modeling, we fitted the structural models of individual NUPs into the cryo–electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaffold. We elucidated in great detail how membrane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane environment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically resolved model covers >90% of the human NPC scaffold. It captures conformational changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dynamic model of the NPC. Our study exemplifies how AI-based structure prediction may accelerate the elucidation of subcellular architecture at atomic resolution. A 70-MDa model of the human nuclear pore complex scaffold architecture. The structural model of the human NPC scaffold is shown for the constricted state as a cut-away view. High-resolution models are color coded according to nucleoporin subcomplex membership. The nuclear envelope is shown as a gray surface. Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport. Their intricate 120-megadalton architecture remains incompletely understood. Here, we report a 70-megadalton model of the human NPC scaffold with explicit membrane and in multiple conformational states. We combined artificial intelligence (AI)–based structure prediction with in situ and in cellulo cryo–electron tomography and integrative modeling. We show that linker nucleoporins spatially organize the scaffold within and across subcomplexes to establish the higher-order structure. Microsecond-long molecular dynamics simulations suggest that the scaffold is not required to stabilize the inner and outer nuclear membrane fusion but rather widens the central pore. Our work exemplifies how AI-based modeling can be integrated with in situ structural biology to understand subcellular architecture across spatial organization levels.