Research Article

Architecture of the symmetric core of the nuclear pore

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Science  15 Apr 2016:
Vol. 352, Issue 6283, aaf1015
DOI: 10.1126/science.aaf1015

Blueprint for a macromolecular machine

Nuclear pore complexes (NPCs) consist of around 1000 protein subunits, are embedded in the membrane that surrounds the nucleus, and regulate transport between the nucleus and the cytoplasm. Although the overall shape of NPCs is known, the details of this macromolecular complex have been obscure. Now, Lin et al. have reconstituted the pore components, determined the interactions between them, and fitted them into a tomographic reconstruction. Kosinski et al. have provided an architectural map of the inner ring of the pore.

Science, this issue pp. 10.1126/science.aaf1015 and 363

Structured Abstract

INTRODUCTION

The nuclear pore complex (NPC) is the primary gateway for the transport of macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and regulator of gene expression. NPCs are very large (~120 MDa) macromolecular machines embedded in the nuclear envelope, each containing ~1000 protein subunits, termed nucleoporins. Despite substantial progress in visualizing the overall shape of the NPC by means of cryoelectron tomography (cryo-ET) and in determining atomic-resolution crystal structures of nucleoporins, the molecular architecture of the assembled NPC has thus far remained poorly understood, hindering the design of mechanistic studies that could investigate its many roles in cell biology.

RATIONALE

Existing cryo-ET reconstructions of the NPC are too low in resolution to allow for de novo structure determination of the NPC or unbiased docking of nucleoporin fragment crystal structures. We sought to bridge this resolution gap by first defining the interaction network of the NPC, focusing on the evolutionarily conserved symmetric core. We developed protocols to reconstitute NPC protomers from purified recombinant proteins, which enabled the generation of a high-resolution biochemical interaction map of the NPC symmetric core. We next determined high-resolution crystal structures of key nucleoporin interactions, providing spatial restraints for their relative orientation. By superposing crystal structures that overlapped in sequence, we generated accurate full-length structures of the large scaffold nucleoporins. Lastly, we used sequential unbiased searches, supported by the biochemical data, to place the nucleoporin crystal structures into a previously determined cryo-ET reconstruction of the intact human NPC, thus generating a composite structure of the entire NPC symmetric core.

RESULTS

Our analysis revealed that the inner and outer rings of the NPC use disparate mechanisms of interaction. Whereas the structured coat nucleoporins of the outer ring form extensive surface contacts, the scaffold proteins of the inner ring are bridged by flexible sequences in linker nucleoporins. Our composite structure revealed a defined spoke architecture in which each of the eight spokes spans the nuclear envelope, with limited cross-spoke interactions. Most nucleoporins are present in 32 copies, with the exceptions of Nup170 and Nup188, which are present in 48 and 16 copies, respectively. Lastly, we observed the arrangement of the channel nucleoporins, which orient their N termini into two 16-membered rings, thus ensuring that their N-terminal FG repeats project evenly into the central transport channel.

CONCLUSION

Our composite structure of the NPC symmetric core can be used as a platform for the rational design of experiments to investigate NPC structure and function. Each nucleoporin occupies multiple distinct biochemical environments, explaining how such a large macromolecular complex can be assembled from a relatively small number of genes. Our integrated, bottom-up approach provides a paradigm for the biochemical and structural characterization of similarly large biological mega-assemblies.

Composite structure of the NPC symmetric core.

The composite structure shown here, viewed from above the cytoplasmic face, was generated by means of sequential unbiased docking of nucleoporin and nucleoporin complex crystal structures into a previously reported cryo-ET reconstruction of the intact human NPC. Nucleoporin structures are shown as colored cartoons, and the nuclear envelope density is shown as a gray surface.

Abstract

The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC’s structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.

The nuclear pore complex (NPC) is a massive molecular transport channel embedded in the nuclear envelope (1). In addition to its role as the sole mediator of bidirectional nucleocytoplasmic transport, the NPC is also involved in diverse cellular processes, including transcription, mRNA maturation, and genome organization (1, 2). Despite their very large size (~120 MDa), NPCs are only composed of 34 different proteins (nucleoporins), which assemble into eightfold-symmetric, ~1000 Å–diameter pores that fuse the inner and outer nuclear membranes (Fig. 1, A and B) (1). Given their central role in cell biology, nucleoporins have been linked to a wide range of human diseases, including viral infection, cancer, and neurodegenerative disease (1, 39). However, the structure of the NPC and the mechanisms by which it influences cellular processes have long been enigmatic.

Fig. 1 Reconstitution of NPC symmetric core protomers.

(A) Cross-sectional conceptual schematic of the NPC. CNCs, colored yellow, form outer rings above the membrane on the nuclear and cytoplasmic faces. Adaptor and channel nucleoporins, respectively colored orange and red, form concentric cylinders in the inner ring. Asymmetric nucleoporins are present on the cytoplasmic and nuclear faces of the NPC. (B) Domain organization of nucleoporins used in biochemical reconstitution experiments. Black lines indicate the construct boundaries used. Domains are drawn as horizontal boxes with residue numbers indicated below and are colored according to the legend (bottom right) for observed or predicted folds. Cartoons to the right are used throughout the figures to represent the nucleoporins. Some nucleoporins form stable complexes [CNC, CFC (cytoplasmic filament nucleoporin complex), and CNT) and are drawn as units. (C) Cartoon of the experimental setup of (D) to (G). The CNC was preincubated with the IRC or Nup188 complex in the presence or absence of Nup145N. Complex formation was only observed in the presence of Nup145N. (D and E) The reconstitution of NPC symmetric core protomers containing the CNC hexamer and IRC is dependent on Nup145N. Identical experiments were performed in (D) the presence or (E) the absence of Nup145N. Size-exclusion chromatography (SEC) coupled to MALS profiles of the complexes in isolation (red and blue) and after preincubation (green) are shown. Colored arrows above the chromatogram indicate the resolved peak fractions. Measured molecular masses are indicated for each peak. Representative Coomassie-stained SDS-PAGE gel slices for the peak fractions are shown in the center. Cartoons below the gel slices illustrate the respective complexes. (F and G) The reconstitution of NPC symmetric core protomers containing the CNC hexamer and Nup188 complex is dependent on Nup145N. Identical experiments were performed in (F) the presence or (G) the absence of Nup145N. Complete SDS-PAGE gels for all panels are shown in fig. S4.

Most nucleoporins are symmetrically distributed in the NPC, forming a symmetric core that in turn recruits asymmetric nucleoporins on the nuclear and cytoplasmic faces (Fig. 1, A and B). Many nucleoporins contain disordered repetitive sequences that are enriched in phenylalanine and glycine residues called FG repeats, which collectively form a central diffusion barrier that prevents passive diffusion of macromolecules with masses greater than ~40 kDa (Fig. 1, A and B). Larger macromolecules can only traffic through the NPC with the assistance of specialized karyopherin transport factors (10).

Despite extensive efforts, the protein-protein interaction network within the NPC has remained incompletely characterized, presenting a fundamental limitation to our understanding of NPC architecture. Copurification and mass spectrometry approaches have revealed some of the strongest interactions, but they provide only general spatial restraints because of their limited resolution. The most well-characterized nucleoporin interactions are those within the coat nucleoporin complex (CNC), which makes up about half the mass of the symmetric core and contains Nup120, Nup85, Sec13, Nup145C, Nup84, Nup133, and, in some species, Seh1, Nup37, Nup43, and ELYS (1). Structural and biochemical analyses of CNCs from multiple species have revealed a highly conserved architecture in which α-helical domains form extensive interaction surfaces to assemble a large Y-shaped complex (1114). Recent advances have dramatically increased the resolution of cryoelectron tomographic (cryo-ET) reconstructions of intact NPCs, facilitating the unbiased placement of 32 copies of a ~400-kDa crystal structure of the yeast CNC into the intact human NPC (13, 14). The CNCs are arranged in pairs of concentric eight-membered rings on both the nuclear and cytoplasmic faces of the NPC, accounting for the majority of the observed protein density in the outer rings of the NPC (13, 14).

Although the organization of the CNC in the intact NPC is well understood, relatively little has been known about the molecular architecture of the inner ring that lines the central channel. The disordered N-terminal FG repeats of the channel nucleoporins Nup49, Nup57, and Nsp1 project into the central channel, and their structured coiled-coil domains form a stable complex, termed the channel nucleoporin heterotrimer (CNT) (15, 16). The CNT, Nic96, Nup192, and Nup145N collectively form a stable subcomplex called the inner ring complex (IRC) (15, 17). The remaining components of the symmetric core, Nup170 and Nup53, are thought to mediate interactions with the nuclear envelope, but the details of the interaction network that assembles these proteins and links them to the CNCs have heretofore been poorly defined (18, 19).

Crystal structures of many nucleoporin fragments from the symmetric NPC core have been determined, including the N-terminal domains (NTDs) of Nup192, Nup188, and Nup157; the C-terminal domain (CTD) of Nup170; the C-terminal tail domains (TAIL) of Nup192 and Nup188; the α-helical solenoid of Nic96, Nic96SOL; and the CNT bound to helical region 1 of Nic96, CNT•Nic96R1 (15, 2026). However, structures of full-length Nup192, Nup188, and Nup170 have remained elusive. Accurate placement of the existing structures into the intact NPC is limited by both their relatively small size and the resolution of available cryo-ET reconstructions. We used an integrated approach to obtain complete atomic structures and accurate, high-resolution biochemical restraints for determining the near-atomic architecture of the NPC symmetric core.

A major barrier preventing complete biochemical characterization of the NPC has been the difficulty of purifying large quantities of full-length nucleoporins from a single species. To overcome this hurdle, we developed expression and purification protocols for all symmetric core nucleoporins from the thermophilic fungus Chaetomium thermophilum, which exhibit superior biochemical stability (27). By reconstituting NPC symmetric core protomers from purified proteins, we were able to determine that the interactions between flexible linker sequences and large scaffold nucleoporins drive NPC assembly. We generated a high-resolution biochemical map for these interactions and determined a series of crystal structures that revealed the structural basis for the recognition of flexible linker sequences by the large scaffold nucleoporins. These crystal structures enabled the construction of complete atomic structures for the ordered scaffolds of Nup170, Nup192, and Nic96. Using our biochemical restraints for validation, we performed unbiased searches to dock the atomic structures into a cryo-ET reconstruction of the intact human NPC with high confidence (28), thus determining a composite structure of the NPC symmetric core.

Reconstitution of NPC symmetric core protomers

Using nucleoporins from C. thermophilum, we first directed our efforts toward reconstituting a soluble protomer that could recapitulate the interaction network within the assembled NPC. We used full-length proteins when possible, but FG repeats, disordered N- or C-terminal regions, or other sequences that prevented soluble protein expression were omitted (Fig. 1B, fig. S1, and tables S1 and S2). We first reconstituted a heterohexameric core CNC containing Nup120, Nup37, ELYS, Nup85, Sec13, and Nup145C, a complex analogous to the yeast CNC that we previously crystallized (fig. S2, A and B) (14). This CNC heterohexamer was assembled with Nup84 and Nup133 to form a heterooctameric CNC (fig. S2C). Because of the poor solubility of the intact heterooctameric CNC, we focused our analysis on its heterohexameric core. Similarly, we extended our previous reconstitution of an IRC containing Nup192, Nic96, Nup145N, and the CNT by also incorporating Nup53 (fig. S3A and table S3) (15). We found that Nup188, which is evolutionarily related to Nup192, failed to incorporate into the IRC (fig. S3B). Rather, an analogous Nup188 complex formed in the absence of Nup192 (Fig. 1F). By preincubating the core CNC hexamer with either the IRC or the Nup188 complex, we reconstituted two distinct 13-protein complexes that are representative of NPC symmetric core protomers (Fig. 1, C, D, and F, and fig. S4, A and C).

Thus equipped with the ability to reconstitute NPC protomers, we sought to identify the interactions that link the IRC or the analogous Nup188 complex to the CNC. Nup145N is a component of the IRC and Nup188 complex, and Nup145C is a component of the CNC. They originate from the same polypeptide chain after posttranslational cleavage mediated by the Nup145N autoproteolytic domain (APD), which is located in the middle of the Nup145 precursor polypeptide (Fig. 1B) (29). Nup145C is composed of a U-bend α-helical solenoid, which is a core component of the CNC, and a disordered ~300-residue N-terminal extension (NTE) (14). Previous studies indicate that Nup145NAPD can still bind the first six N-terminal residues of Nup145C after cleavage, thus offering a possible mechanism for linking the IRC to the CNC (30). The complexes did not interact in the absence of Nup145N (Fig. 1, E and G, and fig. S4, B and D). Moreover, Nup145NAPD alone could be incorporated into the CNC (fig. S5). These results indicate that the IRC and CNC are flexibly attached via the long, intrinsically disordered sequences of Nup145N and Nup145C. Nup145NAPD also binds to the β-propeller domain of the cytoplasmic filament nucleoporin Nup82 (15). However, given that this interaction was outcompeted by CNC binding (fig. S6), another interaction must play a role in retaining the cytoplasmic filaments at the cytoplasmic face of the NPC.

Symmetric core assembly is driven by flexible linkers

We next performed a systematic analysis of the molecular interaction network within the IRC and Nup188 complex, extending our previous work and additionally including Nup170 in our analysis (15). These complexes contain large scaffold domains in Nic96, Nup170, Nup188, and Nup192, as well as long, intrinsically disordered sequences in the linker nucleoporins Nup53 and Nup145N (Fig. 1B). The CNC is primarily held together by extensive interfaces between large α-helical solenoid domains (1, 14), but recent studies have hinted that the architectural principles of the remaining symmetric core nucleoporins are different (15, 17, 27). For example, the scaffold nucleoporin Nic96 possesses a largely unstructured NTE containing two short helical regions, Nic96R1 and Nic96R2, that are essential for IRC assembly: Nic96R1 recruits the CNT to the NPC, and Nic96R2 binds Nup192 or Nup188 (15). To determine whether the structured domains of the scaffold nucleoporins interact with each other to drive symmetric core assembly, we tested whether Nic96SOL, Nup170, Nup192, and Nup188 could form a complex; however, we observed no interaction (Fig. 2A and fig. S7A). Instead, complex formation was achieved only in the presence of the linker nucleoporins Nup53 and Nup145N (Fig. 2, B and C, and fig. S7, B and C). Thus, together with Nic96NTE, the flexible linker nucleoporins Nup53 and Nup145N are the primary driving force of IRC and Nup188 complex assembly (15).

Fig. 2 Biochemical analysis of the interactions mediating NPC symmetric core assembly.

(A) Scaffold domains of Nup192, Nup188, Nup170, and Nic96SOL do not interact with each other. SEC-MALS profiles of the individual scaffolds alone (blue, purple, orange, and green) and after their preincubation with each other (black) are shown. Measured molecular masses are indicated for each peak. (B and C) Linker nucleoporins Nup53 and Nup145N mediate scaffold nucleoporin assembly. SEC-MALS profiles of a scaffold nucleoporin mixture (blue or purple), a linker nucleoporin mixture (red), and these complexes after their preincubation with each other (black) are shown. Mixtures of scaffold nucleoporins contained either (B) Nup192 or (C) Nup188. Complete SDS-PAGE gels for all chromatograms are shown in fig. S7. (D and E) Interaction network between scaffold nucleoporins and (D) Nup53 or (E) Nup145N. Scaffold nucleoporins were tested in SEC-MALS interaction experiments for their ability to form heterodimeric complexes with Nup53 or Nup145N and their compatibility for also forming heterotrimeric complexes. Check marks indicate complexes that can form in SEC-MALS experiments, x’s indicate complexes that do not form, and dashes indicate complexes that were not tested. Complete SEC-MALS chromatograms and SDS-PAGE gels are shown in figs. S8 to S11. (F) Biochemical interaction map determined from SEC-MALS interaction experiments. Minimal regions of Nup145N and Nup53 sufficient for binding to components of the NPC are depicted by colored bars and dashed lines between interacting regions. Interactions that map to the same regions on Nup145N and Nup53 do not occur simultaneously. Complete SEC-MALS chromatograms and SDS-PAGE gels are shown in figs. S13 to S20. The nucleoporin cartoons are according to Fig. 1B.

To further analyze the interaction network between the scaffold and linker nucleoporins, we tested which scaffolds interact with Nup53 or Nup145N to form heterodimers, focusing first on the interactions within the IRC and with Nup170. Nup53 formed robust complexes with Nup192, Nic96SOL, and Nup170 (Fig. 2D and fig. S8). We previously reported that Nup145N interacts weakly with Nic96SOL (15); in this study, we found that Nup145N also binds to Nup192 and Nup170 (Fig. 2E and fig. S9). All of these scaffold-linker interactions are compatible, as demonstrated by the formation of heterotrimeric complexes (Fig. 2, D and E, and figs. S10 and S11). Nup192 and Nup170 can also bind to both linker nucleoporins simultaneously, indicating that the binding sites on the scaffolds are distinct (fig. S12, A and B). We were able to reconstitute a stoichiometric heterotetramer composed of Nup192, Nup170, Nup53, and Nup145N (fig. S12C).

To improve the biochemical resolution of our interaction map, we next identified the minimal sequence fragments of Nup53 and Nup145N that would be sufficient for scaffold recognition. We previously mapped an interaction between a Nup53 fragment, encompassing residues 31 to 67 (Nup53R1), with Nup192NTD (Fig. 2F and fig. S13) (20). In this work, we found an adjacent fragment containing residues 69 to 90 (Nup53R2) that was recognized by Nic96SOL (Fig. 2F and fig. S14, A and B). A Nup53 fragment including both of these binding sites (residues 1 to 90) was sufficient to link the two scaffolds into a heterotrimeric complex (fig. S14C). We also identified a C-terminal Nup53 fragment containing residues 329 to 361 (Nup53R3) that interacted specifically with Nup170NTD (Fig. 2F and fig. S15). Conversely, the association between Nup170 and Nup145N mapped to Nup145N residues 729 to 750 (Nup145NR3) and Nup170CTD (Fig. 2F and fig. S16). Nup192 recognized a fragment of Nup145N that spans residues 606 to 683 (Fig. 2F and fig. S17, A and B). Nup192NTD was sufficient for Nup145N binding (fig. S17C), but we also detected a weak interaction with Nup192CTD (fig. S17, D and E), suggesting that binding sites for Nup145N were distributed throughout Nup192. These minimal sequence fragments were specific for their binding partners (fig. S18).

Whereas preincubation of the two linker nucleoporins with Nup192, Nup170, and Nic96SOL produced a robust pentameric complex, the analogous preincubation with Nup188 in place of Nup192 produced a mixture of species (Fig. 2, B and C, and fig. S7, B and C). To understand this difference in behavior, we repeated the above analysis with Nup188 and identified a robust interaction with Nup145N, whereas Nup53 binding was barely detectible (Fig. 2, D and E, and figs. S8C and S9B). In contrast to our above results, however, Nup188 did not strongly bind Nup145N in the presence of Nup192 or Nup170 (Fig. 2, D and E, and fig. S11). Similar to Nup192NTD, Nup188NTD was sufficient for Nup145N binding (fig. S19, A and B). However, the minimal Nup192-binding fragment of Nup145N was not sufficient for Nup188 binding (fig. S19, D and E). Instead, we only detected robust complex formation with a much longer fragment encompassing both the Nup192- and Nup170-binding sites (residues 606 to 750), which explains the exclusivity of their interactions (Fig. 2F and fig. S19C). We found a similar architecture for the Nup192- and Nup188-binding sites in Nic96R2. The Nup192 minimal binding fragment (residues 286 to 301) again was insufficient for Nup188 binding (fig. S20, A to C), which instead required a larger fragment (residues 274 to 301) (Fig. 2F and fig. S20D). Consistent with these findings, several mutations in the N-terminal region of Nic96R2 ablated Nup188 binding but had no effect on Nup192 binding (fig. S20, E to G) (15). Thus, Nup192 and Nup188 bind competitively to directly overlapping sequences in Nic96 and Nup145N, establishing the existence of a distinct Nup188 complex with an architecture analogous to the IRC.

In summary, we found that interactions between the large ordered scaffold nucleoporins and flexible interaction motifs in Nup53, Nup145N, and Nic96NTE were the dominant driving force for assembly of the NPC symmetric core outside of the CNCs. We built a biochemical map of these interactions by identifying minimal interaction motifs, revealing that the binding sites were spatially distributed throughout the scaffold nucleoporins, but that many of the binding sites on the linker nucleoporins were adjacent or overlapping in sequence (Fig. 2F). In doing so, we identified the exclusive interactions that provide a molecular basis for the formation of two distinct complexes, the Nup192-harboring IRC and an analogous Nup188 complex. Because existing crystal structures have not captured interactions between scaffold and linker nucleoporins, we used these results to identify important structural targets for determining the structural basis for this mode of interaction.

Atomic architecture of the Nup170 interaction network

We determined a crystal structure of the Nup170NTDNup53R3 complex at 2.1 Å resolution (Fig. 3, A and B, and tables S4 and S5). To obtain high-resolution diffraction, we deleted residues 293 to 305 from the 3D4A loop of Nup170NTD (fig. S21). Nup170NTD is composed of a seven-bladed β-propeller and a C-terminal α-helical domain (Fig. 3B). An N-terminal α-helix packs against the C-terminal α-helical domain and is followed by three β-strands that form a triple “Velcro closure” against the β-propeller (Fig. 3B). Nup53 adopts an extended conformation and binds atypically to the side of the β-propeller, rather than the top, at blades 1 and 2 (Fig. 3B). The crystallized Nup53 fragment contained residues 329 to 361, but clear density was only observed for residues 342 to 355. Blade 2 of the Nup170 β-propeller deviates substantially from a canonical β-propeller blade to generate two hydrophobic pockets that accommodate Nup53 residues L346, L347, L353, and L354 (Fig. 3C). We identified several mutations in Nup170 and Nup53 that could disrupt their interaction (Fig. 3, D and E, and fig. S22, A and B). We observed a complete loss of binding with mutations to Nup170 residues that are evolutionary conserved—F199, I203, and Y235—suggesting that the binding interface is evolutionarily conserved (Fig. 3, D and E; fig. S21; and fig. S22, A and B).

Fig. 3 Structural and biochemical analyses of the Nup170 interaction network.

(A) Domain structures of Nup170 and Nup53. Black lines indicate fragments used for crystallization. (B) Cartoon of the crystal structure of the Nup170NTDNup53R3 complex. A 90°-rotated view is shown on the right. The potential WF and ALPS membrane interaction motifs are indicated. (C) Close-up view of the interaction between Nup170 and Nup53. Nup170 and Nup53 residues involved in the interaction are labeled in orange and purple, respectively. (D) Graphic summary of mutational analysis of the Nup170NTD-Nup53R3 interaction. Nup170 is shown in surface representation from the same view as in (C). Residues are colored in red, orange, yellow, or green to indicate mutations that had a strong, moderate, weak, or nonexistent effect on binding, respectively. (E) Tabular summary of tested Nup170 mutants and their effect on Nup53 binding. Three plus signs indicate wild-type binding, two plus signs indicate moderately weakened binding, one plus sign indicates weak binding, and a dash indicates no binding. Mutations in Nup53 that were tested for binding are indicated by dots above the sequence, following the same color code as in (D). Chromatograms and representative SDS-PAGE gels are shown in fig. S22. (F) Domain structures of Nup170 and Nup145N. Black lines indicate fragments used for crystallization. (G) Cartoon of the crystal structure of the Nup170CTDNup145NR3 complex. (H) Close-up view of the interaction between Nup170 and Nup145N. Nup170 and Nup145N residues involved in the interaction are labeled in orange and cyan, respectively. (I) Graphic summary of mutational analysis of the Nup170CTD-Nup145NR3 interaction. Nup170 is shown in surface representation from the same view as in (H). Coloring is according to (D). (J) Tabular summary of mutational analysis of the Nup170CTD-Nup145NR3 interaction, with the same symbols and coloring as in (E). Chromatograms and representative SDS-PAGE gels are shown in fig. S23. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Nup53 is anchored to the nuclear envelope by its C-terminal amphipathic helix, either directly or through an interaction with NDC1 (18, 19). We found that Nup170 binds to Nup53R3, which is directly adjacent to this C-terminal helix, prompting us to look for features in Nup170 that could also contribute to nuclear envelope binding. We identified two motifs next to the C terminus of Nup53R3 that would be juxtaposed with the nuclear envelope. The first was a WF motif composed of solvent-exposed, evolutionarily conserved tryptophan and phenylalanine residues in the 3CD loop (Fig. 3B and fig. S22C). Because tryptophan residues are enriched at membrane interfaces, the WF motif may reinforce membrane binding (31). The second motif, residing in the 3D4A loop that we deleted for crystallization, is predicted to form an amphipathic helix with an evolutionarily conserved absence of charged residues; this feature is characteristic of amphipathic lipid packing sensing (ALPS) motifs, which are also present in Nup120 and Nup133 (fig. S22, C and D) (32, 33). The Nup170 ALPS motif contains a universally conserved proline residue on the polar face of the helix, a feature reminiscent of antimicrobial membrane destabilizing peptides (fig. S22D) (34). We propose that these additional features on Nup170 act synergistically with the binding of Nup53 to the nuclear envelope to help maintain the extreme membrane curvature in nuclear pores.

We next determined a crystal structure of the Nup170CTDNup145NR3 complex at 3.5 Å resolution, using a 2.1 Å–resolution structure of Nup170CTD in the apo state as a search model (Fig. 3, F and G, and tables S4 and S6). Nup170CTD forms an elongated α-helical solenoid containing two stacks of irregular helical pairs, arranged in a zig-zag fashion (Fig. 3G). The two stacks share a long helix (α31) that caps the first stack and initiates the second stack. Nup145NR3 binds to a pair of deep hydrophobic pockets on either side of the first two helices of the second helical stack, inserting residues L733 and I735 into the first pocket and L743 and F744 into the second pocket (Fig. 3, G and H). Mutation of any of these residues completely abolished binding to Nup170 (Fig. 3, I and J, and fig. S23B). Similarly, mutation of the residues that formed the hydrophobic pockets (F1171, F1154, I1131, and Y1157) strongly affected binding (Fig. 3, I and J, and fig. S23A). The hydrophobic nature of both binding pockets is retained throughout eukaryotes (fig. S21), suggesting the evolutionary conservation of this interaction. Only minimal rearrangements of the binding pocket occur upon Nup145N binding (fig. S23C).

The Nup145N sequence that binds to Nup170 is also highly conserved throughout eukaryotes, and the homologous residues that are critical for Nup170 binding are conserved in humans (fig. S24A). During mitosis, extensive phosphorylation of hsNup98 (hs, Homo sapiens), the human homolog of Nup145N, leads to NPC and nuclear envelope disassembly (35). The most abundant mitotic phosphorylation sites in hsNup98 are at residues S608 and S612 (S741 and S745 in C. thermophilum), which flank L610 and F611 (L743 and F744 in C. thermophilum—residues that we found to be critical for Nup170 binding; fig. S24, A and B) (35). To test the possibility that the interaction between Nup170 and Nup145N could be regulated by phosphorylation, we reconstituted a hsNup155hsNup98 heterodimer that was homologous to our crystallized complex. We observed a robust interaction between hsNup155CTD and the corresponding minimal hsNup98 fragment, which was partially disrupted by a double phosphomimetic mutation (S608E and S612E) (fig. S24C). As revealed by the Nup170CTDNup145NR3 structure, S612 (S745 in C. thermophilum) forms a hydrogen bond with N609 (D743 in C. thermophilum). Phosphorylation would therefore destabilize the conformation required to insert the critical hydrophobic residues into the binding pocket in Nup170 (fig. S24B). Disruption of this hydrogen bond by mutagenesis also completely abolished binding (Fig. 3J and fig. S23B). Thus, the Nup170-Nup145N interaction is not only highly conserved, but its disruption is also a key step in mitotic NPC disassembly in humans.

Our structures of Nup170NTD and Nup170CTD did not overlap in sequence, preventing accurate modeling of the full-length protein. Therefore, we crystallized a larger fragment of Nup170 that contained the α-helical solenoids of both domains (Nup170SOL, residues 575 to 1402), and determined the crystal structure at 4.0 Å resolution (fig. S25A, tables S4 and S5, and Movie 1). Whereas we observed no conformational variability for the helices present in Nup170NTD, the Nup170CTD solenoid exhibited a ~20° rotation resulting from a minor rearrangement of helix α27 (fig. S25A). We observed a similar conformational variability in the Nup170CTDNup145NR3 complex structure, where all four molecules in the asymmetric unit adopted different conformations (fig. S25B). With the structure of Nup170SOL as a template, we superposed the structures of Nup170NTD and Nup170CTD, obtaining a total of eight different conformations for full-length Nup170 (figs. S25B and S26 and Movie 2).

Movie 1 Generation of the Nup170•Nup53R3•Nup145NR3 complex structure by superposition.

A 360° rotation of the crystal structure of Nup170SOL is shown, followed by a demonstration of the process used for generating the full-length structure by superposition. Colors are the same as in fig. S25.

Movie 2 Morph animation of observed Nup170 conformations.

The eight different conformations observed in the superposition-generated structures of full-length Nup170 are animated. The animation pauses at each of the eight experimentally observed conformations of Nup170, which are colored the same as in fig. S25.

Molecular basis for recognition of Nup53 by Nic96

We determined crystal structures of apo Nic96SOL and a Nic96SOLNup53R2 complex at 3.3 and 2.65 Å resolution, respectively (Fig. 4, A and B; fig. S27; tables S4 and S7; and Movie 3). Nic96SOL forms a rod-shaped molecule consisting of a U-bend α-helical solenoid with the N terminus situated in the middle of the rod. Although residues 31 to 84 of Nup53 were included in the crystallization construct, only residues 67 to 84 were visible in the electron density. Residues 67 to 84 of Nup53 form an amphipathic helix that buries its hydrophobic face into a hydrophobic groove formed by helices α14, α15, and α16 near the U-bend end of the Nic96 solenoid (Fig. 4, B and C). Consistent with our crystal structure, we found that mutations to the hydrophobic residues in this pocket disrupted binding (figs. S28 and S29). Similar to the interaction between Nup170 and Nup145N, we observed minimal conformational rearrangements upon Nup53 binding (Fig. 4D).

Fig. 4 Structural analysis of the IRC nucleoporins Nic96 and Nup192.

(A) Domain structures of Nic96 and Nup53. Black lines indicate fragments used for crystallization. (B) Cartoons of the crystal structures of apo Nic96SOL (yellow), the Nic96SOLNup53R2 complex (green and purple), and their superposition (see also Movie 3). (C) Close-up view of the Nup53R2-binding site in Nic96SOL. For clarity, Nup53 is shown in ribbon representation. Nic96 and Nup53 residues involved in the interaction are labeled in green and purple, respectively. (D) Close-up view of the superposition of apo and Nup53-bound structures of Nic96SOL, revealing minimal conformational changes. (E) Domain structure of Nup192. The black line indicates the fragment used for crystallization. (F) Cartoon of the crystal structure of Nup192ΔHEAD. A 180°-rotated view is shown on the right. Regions of Nup192 that were resolved in previous crystal structures are colored in shades of blue; the region of the protein that was not included in previous crystallographic analyses is shaded cyan. (G) Cartoon of the structure of full-length Nup192, generated by superposing fragment crystal structures (see also fig. S31 and Movie 4).

Movie 3 Structure of the Nic96SOL•Nup53R2 complex.

A 360° rotation of the crystal structure of Nic96SOL •Nup53R2 is shown. Colors are the same as in Fig. 4.

Structure of Nup192

Nup192, the largest symmetric core nucleoporin, appears to be shaped like a question-mark at low resolution (26). Crystal structures exist for Nup192NTD (residues 1 to 958) and Nup192TAIL (residues 1397 to 1756), but the atomic structure of the entire molecule has thus far remained unresolved (15, 20, 26). To determine the complete atomic structure of Nup192, we obtained crystals of an engineered Nup192 truncation mutant, Nup192ΔHEAD, from which we had deleted (Δ) the N-terminal head domain (HEAD; residues 1 to 152) and replaced a loop encompassing residues 167 to 184 with a short glycine-serine linker. We determined the crystal structure of Nup192ΔHEAD at 3.2 Å resolution (Fig. 4, E and F; fig. S30; and tables S4 and S7).

The N-terminal portion of the previously unresolved middle domain of Nup192 (Nup192MID) contains three additional ARM repeats (α46 to α53, residues 959 to 1154) that continue the superhelical solenoid that we previously observed in Nup192NTD (Fig. 4F) (20). Similarly, the C-terminal portion of Nup192MID contains a HEAT repeat (α60 to α61, residues 1330 to 1376) that extends the Nup192TAIL solenoid, so that the entire protein forms a continuous HEAT-ARM repeat solenoid (Fig. 4F) (15). However, we observed an unusual insertion (residues 1155 to 1329) between the ARM repeats and the HEAT repeat in Nup192MID, containing a ~50-residue helix, α58, that reaches ~75 Å from the beginning of Nup192TAIL to the C terminus of Nup192NTD (Fig. 4F). This insertion, which we termed the Tower helix, buries several hydrophobic residues against the bottom of Nup192NTD, inducing minor rearrangements that facilitate packing of the Tower helix. Although the Tower helix has only a moderate signature in secondary structure predictions, the evolutionarily related Nup188 is also predicted to contain a similarly long ~40-residue helix at the same location. However, previous models of full-length Nup188 and Nup192 did not anticipate the existence of the Tower helix, highlighting the importance of experimentally determining atomic-resolution structures (22, 36).

Taking advantage of the extensive overlap between our crystal structure of Nup192ΔHEAD and the existing structures of Nup192NTD and Nup192TAIL, we generated the structure of full-length Nup192 by superposition (Fig. 4G, fig S31, and Movie 4). Inspection of the full-length protein revealed that the first loop in the HEAD domain between α1 and α2 is close enough to contact loops in the MID domain, predominantly with polar and charged residues (Fig. 4G and fig. S31). The binding sites on Nup192 for Nup53 and Nic96, which we previously identified via mutagenesis, are respectively located at the top and bottom of the molecule, ~140 Å apart from each other (fig. S32) (15, 20).

Movie 4 Generation of the structure of full-length Nup192 by superposition.

A 360° rotation of the crystal structure of Nup192∆HEAD is shown, followed by a demonstration of the process used for generating the full-length structure by superposition. Colors are the same as in fig. S31.

Architecture of the NPC symmetric core

Recent advances in cryo-ET have produced rapidly improving reconstructions of intact NPCs, with the most recent reconstructions reporting average resolutions up to ~20 Å in the best-resolved regions (13, 28, 37). We previously docked 32 copies of the yeast CNC into a ~34 Å reconstruction of the intact human NPC, taking advantage of the distinctive shape and large size of the complex (14). With the addition of the Nic96SOLNup53R2 structure and the full-length superposition-generated structures of Nup192 and Nup170Nup53R3Nup145NR3 reported here, as well as our recently reported crystal structure of the CNTNic96R1 complex, accurate structures were available for essentially the entire ordered mass of the NPC symmetric core (15). The domain architectures of the symmetric core nucleoporins are highly conserved from fungi to humans (fig. S33). We successfully located these structures in the recently reported ~23 Å reconstruction of the human NPC, and the arrangement of nucleoporins was validated by our biochemical restraints (28). We used an incremental approach to confidently place the crystal structures, starting with the largest structures with the most distinctive shapes and iteratively removing the occupied density to search for other structures (fig. S34). Because the cryo-ET map possesses eightfold rotational symmetry, each solution defined the location and orientation of eight copies of each molecule. We first tested our approach with the yeast CNC crystal structure and found four distinct placements with exceptional scores compared with 50,000 other refined placements, which is in excellent agreement with our previous results (fig. S35A) (14). We also readily identified the location and orientation of human Nup84CTDNup133CTD in unbiased searches (fig. S35B) (38). Based on previously reported biochemical data, we manually docked the Nup37, Nup43, and Nup133 β-propellers and locally optimized their fit (fig. S35C) (13, 3941).

We next performed unbiased searches for Nup170 and Nup192 by using a map from which any density corresponding to the CNCs had been removed (fig. S36). Because our crystallographic data indicated considerable flexibility in the Nup170 solenoid, we performed searches with the eight different conformations of Nup170. These searches identified two conformations that each yielded two distinct top-scoring solutions (fig. S36, A and B). Searches with full-length Nup192 revealed six solutions, but because Nup192 and Nup188 could not be distinguished at this resolution, we assigned two of these as Nup188 based on our biochemical results and previously reported cross-linking data, as detailed in the methods (fig. S36C) (13, 42). After removing the density assigned to Nup170, Nup192, and Nup188, we successfully located four distinct copies of the Nic96SOL•Nup53R2 and CNT•Nic96R1 complexes (fig. S37, A and B). Lastly, we inspected the remaining density in the inner ring in an attempt to locate the ordered domains of Nup53 and Nup145N. We determined crystal structures of Nup53RRM (RRM, RNA recognition motif domain) and Nup145NAPD•Nup145CN (where the superscript N denotes residues 1 to 6) at 0.8 and 1.3 Å resolution, respectively, but we could not unambiguously place them because of their small size and globular shape (fig. S38 and tables S4, S8, and S9). We attempted to generate biochemical restraints to confidently dock Nup53RRM, but we were unable to find any binding partners (fig. S39). However, we did find a pair of continuous densities that readily accommodated two additional Nup170 molecules in a third distinct conformation (fig. S40A). These placements were buried in our original global search, but the conformation of this Nup170 structure nevertheless differed slightly from the remaining map density, suggesting that our crystal structures did not capture the full conformational range of Nup170 (fig. S40, B and C).

Having placed the structures of the CNC hexamer, Nup84CTD•Nup133CTD, Nup133NTD, Nup37NTD, Nup43, Nup188NTD, Nup188TAIL, Nup192, Nup170•Nup53R3•Nup145NR3, Nic96SOL•Nup53R2, and CNT•Nic96R1, we acquired a composite structure that accounts for nearly all of the density in the NPC symmetric core, corresponding to ~56 MDa of protein mass, or ~320,000 ordered residues (Fig. 5, fig. S41, table S10, and Movie 5). In total, the composite structure contains a stoichiometry of 16 copies of Nup188; 32 copies of the CNC, Nup192, Nic96, and CNT; and 48 copies of Nup170, Nup53, and Nup145N. Overall, this stoichiometry is in good agreement with a previous study that used mass spectrometry to measure the relative abundances of nucleoporins in the human NPC (43).

Fig. 5 Architecture of the NPC symmetric core.

The composite structure of the NPC symmetric core was generated by docking nucleoporin and nucleoporin complex crystal structures into the cryo-ET reconstruction of the intact human NPC (Electron Microscopy Data Bank entry number EMD-3103). The density corresponding to the nuclear envelope is shown as a gray surface. Proteins are color-coded according to the legend at the bottom. (A) View from above the cytoplasmic face and (B) a cross-sectional view from within the transport channel (see also figs. S34 to S37 and S40 and Movie 5).

Movie 5 Overview of the composite structure of the NPC symmetric core.

The structures are shown as docked into the cryo-ET reconstruction of the intact human NPC, oriented with the cytoplasmic face on top and the nuclear face on the bottom. The nuclear envelope and protein densities are colored in gray and white, respectively. Crystal structures of symmetric core nucleoporins are first shown as cartoons (colored according to Fig. 5), then without the cryo-ET reconstruction density, and finally in surface representation.

Spoke architecture

The symmetric core of the NPC consists of eight spokes related by an eightfold rotational axis of symmetry that is perpendicular to the nuclear envelope (Fig. 5A). Outer rings reside above the nuclear and cytoplasmic faces of the nuclear envelope, and an inner ring is embedded in the pore and spans the nuclear envelope (Fig. 5B). When viewed from the cytoplasm, the nucleoporins form distinct cylinders, with the CNTs lining the transport channel, surrounded by successive cylinders formed by Nup192, Nic96, Nup170, and the CNCs (Fig. 5A). Nic96NTE and the linker nucleoporins Nup53 and Nup145N span these cylinders. Only C8 rotational symmetry was applied to generate the cryo-ET reconstruction, yet we observed an additional twofold axis of symmetry in our composite structure that relates the nuclear and cytoplasmic sides within each spoke (Fig. 6, A to D) (28).

Fig. 6 Architecture of the NPC spoke.

(A) A cartoon of a single spoke of the NPC symmetric core is shown from the same cross-sectional view as in Fig. 5B. Different shades of colors are used to indicate biochemically distinct dockings of the same protein (see also Movie 6). (B) The same view as in (A), but with Nup170 and the nucleoporins from the outer ring removed to highlight the organization of the four IRCs. (C) The membrane coat of the NPC. Nup192 and Nic96 molecules and CNTs have been removed for clarity. Contacts between the nuclear envelope and the ALPS motifs in Nup120, Nup133, and Nup170 are indicated by dots (numbered as a guide to the eye). (D) Schematic of the NPC spoke. The proteins corresponding to the nuclear side of the spoke are colored in gray, demonstrating the twofold rotational symmetry relating the cytoplasmic and nuclear halves of each spoke. (E) Equatorial and peripheral IRCs adopt similar conformations. Identical views of the cytoplasmic peripheral IRC, the cytoplasmic equatorial IRCs, and their superposition are shown. For clarity, the equatorial IRC is colored in gray in the superposition. (F) Organization of FG repeats in the inner ring. Colored spheres indicate the positions of the N termini of the three channel nucleoporins for all 32 CNT copies in the inner ring. Despite four distinct CNT positions in each spoke, the N termini for the cytoplasmic and nuclear complexes are arranged in approximately the same plane. Thus, though not possessing true 16-fold symmetry, the CNT N termini approximately form two 16-membered rings, which are indicated by a solid and a dashed line. The FG repeats of the 16-membered CNT rings project circumferentially into the central transport channel with opposite directionality (cytoplasmic CNT ring, counterclockwise; nuclear CNT ring, clockwise). Cartoons of the CNT molecules for a single spoke are shown to indicate the directionality of the FG repeats.

Our results provided spatial restraints for Nic96R1 and Nic96R2, allowing us to trace the path of Nic96NTE, which emerges from the middle of Nic96SOL, to its binding sites on Nup192 and the CNT. Each inner ring spoke contains four copies of the IRC, which we defined based on this connectivity (Fig. 6B and Movie 6). Here we refer to these four distinct IRCs as nuclear peripheral, nuclear equatorial, cytoplasmic peripheral, and cytoplasmic equatorial IRCs (Fig. 6B). The nuclear and cytoplasmic equatorial IRCs are related to each other directly by the twofold rotational axis of symmetry, as are the nuclear and cytoplasmic peripheral IRCs. Unexpectedly, the subunits in the equatorial and peripheral IRCs are in approximately the same relative orientation, which was readily apparent upon superposition (Fig. 6E). Because the subunits were placed independently, this unexpected symmetry is an emergent property of the composite structure. In the composite structure, the CNT and Nup192 are in close proximity, suggesting that additional weaker interactions orient the CNTs in the fully assembled NPC. We observed additional knobs of density adjacent to the Nup57 α/β domains of each CNT, which were unexplained by our composite structure (fig. S37D). When we superposed structures of CNT fragments from Xenopus laevis onto our docked CNT molecules (16), we found that the metazoan-specific ferredoxin-like domain of Nup57 accounts for these extra knobs of density (fig. S37, C and D), further validating our composite structure.

Movie 6 Rotating structure of a single spoke of the NPC symmetric core.

The crystal structures are shown as docked into the cryo-ET reconstruction of the intact human NPC, oriented with the cytoplasmic face on top and the nuclear face on the bottom. The nuclear envelope is colored in gray. Crystal structures of symmetric core nucleoporins are shown as cartoons colored according to Fig. 6.

The mechanism by which FG repeats in the central transport channel form a diffusion barrier and facilitate transport is controversial, partly because the stoichiometry and orientation of the FG repeats were previously unknown. Our composite structure revealed a total of 32 CNTs in the inner ring, which would project 96 distinct polypeptide chains in clusters of three into the central channel (Fig. 6F). The orientation of the CNTs suggests that the FG repeats would emanate circumferentially toward the adjacent spoke, rather than pointing radially toward the center of the channel. Unexpectedly, the N termini of the peripheral and equatorial CNTs were evenly spaced and roughly planar, so that they approximately formed two 16-membered rings (Fig. 6F).

In the outer rings, each spoke contains two CNCs on either face, which we refer to as proximal and distal CNCs, based on their distance from the inner ring. The orientations of Nup133 relative to the CNC core differ slightly between the distal and proximal CNCs but create the same overall architecture: Each pair of distal and proximal CNCs forms an arch over the nuclear envelope (fig. S42). The outer rings also contain a Nup188 molecule on either face (fig. S43). The majority of the CNC components are ~100 Å above the membrane, and the only contacts with the membrane are made by the β-propeller domains of Nup120 and Nup133 through their ALPS motifs (Fig. 6C). Similarly, the IRCs do not make direct contacts with the membrane but instead are surrounded by a network of Nup170 molecules that form the outermost layer of the inner ring (Fig. 6C). Each spoke contains three distinct pairs of Nup170 molecules, which we refer to as equatorial, peripheral, and bridging Nup170 molecules. The equatorial pair occupies alternating orientations equatorially along the surface of the nuclear envelope (Fig. 6C and fig. S36A). The resolution of the cryo-ET reconstruction of these molecules is high enough that the central holes of the β-propellers are readily visible at higher contour levels (fig. S36A). The bridging pair of Nup170 molecules connect the inner ring to the outer CNC rings through a contact with Nup120 (Fig. 6C and figs. S36B and S43). The peripheral Nup170 molecules, which have a weaker quality of fit and were identified only after placing all other symmetric core components, contact both the equatorial and bridging Nup170 molecules (Fig. 6C and fig. S40). The equatorial Nic96 molecules contact multiple Nup170 molecules, effectively bridging the nuclear and cytoplasmic networks. The ALPS and WF motifs that we identified in Nup170NTD and the C-terminal amphipathic helix of Nup53 are positioned directly adjacent to the nuclear envelope (Fig. 6C). Thus, Nup170 and Nic96 constitute a membrane coat for the inner ring that is analogous to the CNCs in the outer ring.

Inter-spoke interactions

We next searched for interfaces that mediate interactions between spokes. Several potential interactions have been identified in previous studies, including one between Nup133NTE and Nup120NTD (40). Our composite structure revealed four additional interactions between CNC components that could link adjacent spokes: (i) between the neighboring proximal Nup84 and the distal Nup85, (ii) between the proximal Nup133 α-helical solenoid and the distal Nup120 α-helical solenoid, (iii) between the proximal Nup133 β-propeller and the proximal Nup120 β-propeller, and (iv) between the distal Nup133 β-propeller and the distal Nup120 β-propeller (fig. S43, A to C). The space between the proximal and distal CNCs contains a density that readily accommodates a Nup188 molecule. Nup188 recognizes a special niche generated in the CNC inter-spoke interface, bridging four CNC molecules by potentially making contacts with (v) the distal Sec13, (vi) the distal Nup85, (vii) the proximal Nup43, and (viii) the proximal Nup133 of a neighboring spoke (fig. S43, B and C). The Nic96R2-binding site in Nup188TAIL is not occluded by any additional density (fig. S43B). Because of the absence of strong density in this region, we cannot determine whether only Nup188 or an entire Nup188 complex would be anchored to the outer rings at this site. However, any flexibly tethered components of the NPC, including the remainder of the Nup188 complex, may not be clearly visible in cryo-ET reconstructions.

Inspection of the interaction surfaces also provided a molecular explanation for how two CNC rings are assembled. Nup133 and Nup170 bind to distinct Nup120 molecules via overlapping interfaces on the proximal and distal Nup120 molecules, respectively, effectively capping the CNCs on either side (fig. S43, D and E). These results are in agreement with a common evolutionary origin for Nup120, Nup133, and Nup170, which all possess similar domain architectures, contact the nuclear envelope via ALPS motifs, and interact with each other at inter-spoke interfaces. The U-bend solenoid nucleoporins—Nup85, Nup145C, Nup84, and Nic96—bridge these inter-spoke interfaces to form a continuous membrane-bending coat. These results also support the protocoatomer hypothesis of a common evolutionary origin for vesicle coats and the NPC coat, which contends that the extant nucleoporins derive from an ancient membrane coat that contained these protein folds (44).

We could only identify a single interaction that would analogously link the inner ring spokes, which would be mediated by an interaction between Nup53R1 and Nup192 (Fig. 7). In our composite structure, peripheral Nic96 molecules orient Nup53R2 directly adjacent to the Nup53-binding site on equatorial Nup192 molecules from a neighboring spoke (Fig. 7B). Our biochemical mapping experiments identified adjacent binding sites for Nic96 and Nup192 on Nup53 (Fig. 2F) and indicate that a fragment containing both of these binding sites could bridge the two nucleoporins (fig. S14C). Thus, binding of Nup53R1 in trans to a Nup192 molecule from a neighboring spoke would link the inner ring spokes.

Fig. 7 Cylindrical organization of the NPC.

(A) Top view of the composite structure of the NPC symmetric core, viewed from the cytoplasm (left). Coloring is according to Fig. 5. The schematic representation on the right illustrates the distinct concentric cylinders observed in the symmetric core. Cartoons of the linker nucleoporins and the unstructured NTE of Nic96 are shown to indicate how they span across multiple cylinders. Arrows indicate gaps between the inner ring spokes, which represent proposed paths for inner nuclear membrane protein transport. (B) Only a single contact is observed between two adjacent inner ring spokes. A close-up view of the inter-spoke interface in the inner ring is shown in an orientation similar to Fig. 5A. The two adjacent spokes are depicted as gray and white surfaces on the left and right, respectively. An equatorial Nup192 molecule (spoke 1) and a peripheral Nic96 molecule bound to Nup53 (spoke 2) are shown as cartoons. The Nup53-binding site on Nup192 is labeled, and the proposed path of the peptide is drawn as a purple dashed line. (C) Schematic illustrating the concentric cylinder organization of the symmetric core, viewed from the side. The order and arrangement of the binding sites for the linker nucleoporins observed in the composite structure are depicted. The proposed inter-spoke interaction between Nup53 and Nup192 is drawn at the bottom.

An open question regarding nucleocytoplasmic transport has been the mechanism of inner nuclear membrane (INM) protein transport through the NPC, particularly for INM proteins with large globular nuclear domains. Peripheral channels on the order of ~100 Å have been proposed as routes for INM transport (45), but we observed no such channels through the inner ring in either our composite structure or the cryo-ET reconstruction (Fig. 5). Given the dense packing within each spoke, traffic of INM proteins through a spoke would require considerable disruption of the NPC structure (Fig. 7A). Rather, the most likely path through the inner ring would be at the inter-spoke interfaces where Nup53R1 and Nup192 interact (Fig. 7). The CNCs form a ~100 Å arch above this interface, providing an uninterrupted path to the inner ring and possibly explaining the previously observed upper limit for the size of nuclear domains (Fig. 6A) (46). However, the channel at the inner ring is much smaller, suggesting that rearrangements at the inter-spoke interface may be necessary to traffic large nuclear domains. Thus, our composite structure of the NPC symmetric core enables rational design of experiments to further understand the mechanism of INM protein import.

A flexible linker mediates CNC oligomerization

While reconstituting the CNCs, we noticed that the assembly of the CNC octamer spontaneously generated a separate solution phase (fig. S44A). Similar phase transitions have previously been observed in other systems with multiple binding valencies, suggesting that the oil droplet formation that we observed resulted from oligomerization of the CNC (47). We previously identified an interaction between Nup133NTE and Nup120 that would mediate head-to-tail CNC ring formation, which would be consistent with the composite structure of the NPC (40). Removal of the unstructured Nup133NTE completely ablated both oil droplet and complex formation, suggesting that this flexible interaction serves as a driving force for CNC ring formation (fig. S44). We also found that Nup170 could be incorporated into this separate solution phase and that this incorporation was ablated by C-terminal truncation, which is consistent with the interaction between the proximal Nup120 and bridging Nup170 molecules that we observed in our composite structure (fig. S44A).

Conservation of NPC architecture

Our results establish the principles that drive the assembly of nucleoporins in the NPC. Whereas the outer rings assemble largely through structurally rigid interaction surfaces, inner ring assembly is primarily driven by flexible linker sequences within Nup53, Nup145N, and Nic96NTE. This dichotomy may reflect the different roles of the respective complexes. The outer rings provide a structural scaffold for the NPC, and, given their location above the plane of the nuclear envelope, their assembly would not be greatly affected by the dynamic generation of membrane curvature during fusion of the inner and outer nuclear membranes. In contrast, the proteins in the inner ring occupy an environment that only exists after membrane fusion, probably necessitating conformational flexibility over the course of NPC assembly.

The importance of these flexible interactions in the NPC is highlighted by their evolutionary conservation, despite poor overall sequence conservation in the linker nucleoporins Nup53 and Nup145N. The overall folds of the scaffold proteins are well conserved in S. cerevisiae (sc) (fig. S45); furthermore, point mutations in the binding pockets of scNic96, scNup170, and scNup157 disrupted their interaction with linker sequences (figs. S46 to S48). A complete understanding of the interaction network in S. cerevisiae has been difficult to obtain because of the genetic redundancy that arises from several gene duplications (Nup170 and Nup157; Nup53 and Nup59; and Nup145N, Nup100, and Nup116). We found that the paralogs mostly retained the ability to form these interactions, but we did not detect an interaction between scNup188 and any of the Nup145N paralogs (figs. S46 to S50).

Our structural data also highlight the evolutionary conservation of the structure and interactions of nucleoporins. Although crystal structures of the human scaffold nucleoporins have not been determined, previous comparisons of the fungal CNC with low-resolution reconstructions of the human CNC suggest a conserved architecture (13, 14). Superposition of the structures of Nup53RRM and Nup145NAPD with their human homologs also revealed that their folds are identical (fig. S38) (30). In addition, the mechanism of interaction between Nup170 and Nup145N is conserved in humans, and we found that phosphomimetic mutations weakened the interaction between hsNup98 and hsNup155. Several other phosphorylation sites have been identified in Nup98, many of which potentially overlap with scaffold binding sites (35). Therefore, phosphorylation could also regulate other key interactions, including those that occur between Nup145N and Nup188, Nup192, and the CNC. Nup53 is similarly phosphorylated in a cell cycle–dependent manner, and phosphorylation of both linker nucleoporins thus would be an effective means to disassemble the entire inner ring of the NPC (48).

Conclusions

Determining the molecular details of nucleocytoplasmic transport has been a longstanding challenge, at least in part due to an incomplete understanding of the architecture and biochemistry of the NPC itself. We used purified recombinant proteins to systematically characterize the nucleoporin interaction network and determine atomic-resolution structures of nucleoporin complexes. This approach was crucially complemented by recent advances in cryo-ET reconstructions (28). Using the results of our divide-and-conquer approach, we were able to dock the available crystal structures into a cryo-ET reconstruction of the human NPC, yielding a composite structure for the entire NPC symmetric core. This union of bottom-up and top-down approaches offers a paradigm for determining the architectures of similarly complex macromolecular assemblies.

Our composite structure differs dramatically from previously reported computational models, not only in relative and absolute stoichiometry but also in overall architecture (49). These discrepancies highlight the complexities that must be accommodated when attempting a holistic, computational approach. We observed a high degree of symmetry in the structure of the NPC, which explains how such a limited vocabulary of proteins can generate such a large macromolecular structure. Most nucleoporins also occupy multiple distinct biochemical environments. Nup170 offers a dramatic example of this property: Biochemically distinct versions of Nup170 are either buried in the inner ring or are exposed in the bridge between the inner and outer rings. Similarly, Nup120 uses overlapping, exclusive interfaces to contact Nup170 and Nup133. Because of this diversification of nucleoporin function, the NPC can be encoded by a relatively small number of genes. The gene duplications of nucleoporins in S. cerevisiae may reflect the gradual separation of these distinct functions into several genes. Nup170 appears to also adopt different conformations in each of its distinct biochemical environments, which is consistent with the wide conformational range that we observed in the crystal structures. It is possible that the different conformations are the result of different mechanical forces acting on Nup170 at each position.

Biochemical diversification of proteins within the same protein complex also generates enormous challenges for computationally modeling the structure of the NPC and similar complexes, because distance restraints such as cross-links that are valid in one biochemical environment may be violated in another. This challenge is exacerbated by the possibility of flexibly tethered domains or nucleoporins, such as those in the Nup188 complex. Our results also highlight the confounding effect generated by the flexible linker nucleoporins Nup53 and Nup145N, which occupy binding sites that span the entirety of the inner ring, rather than a single globular volume. The structure of the NPC could be used as a template for the development of methods that can accommodate these additional complexities.

Our composite structure of the NPC provides a rich platform for contextualizing previous results, not all of which can be discussed here. The structure also permits the rational design of new experiments to not only further validate the structure, but also to begin structure-function investigation of the NPC. Our biochemical results led to a map of the strongest and most conserved interactions, but our composite structure indicates that many additional interactions can occur in the context of the assembled NPC. However, a structural understanding of the entire NPC at single-residue resolution still requires several advances. Successful placement of crystal structures of fungal nucleoporins into a cryo-ET reconstruction of the intact human NPC demonstrates not only the evolutionary conservation of NPC structure, but also the need for structural characterization of human nucleoporins and further improvement of the resolution of cryo-ET reconstructions to increase the accuracy of the composite structure of the NPC. Improved resolution in cryo-ET reconstructions in which secondary structure elements can be visualized would enable flexible fitting of high-resolution crystal structures. We were not able to dock any of the asymmetric components of NPC into the composite structure, because of the small size of extant structures and the absence of high-quality restraints (fig. S41). A similar approach to the one used here will be necessary to extend our analysis to the many proteins in the cytoplasmic filaments and nuclear basket that interact directly with transport factors and that are implicated in human disease. Lastly, high-resolution structural characterization of the remaining interactions, especially those involving flexible linker sequences, remains critical to building a truly complete structure of the NPC, because these interactions may never be resolved in cryo-ET reconstructions.

Materials and methods

Bacterial expression constructs

DNA fragments were amplified by polymerase chain reaction using C. thermophilum and H. sapiens cDNA. SUMO (small ubiquitin-like modifier)–tagged proteins were cloned into a modified pET28a or pET-MCN vector containing an N-terminal hexahistidine tag followed by a SUMO tag by using BamHI and NotI restriction sites (50, 51). Hexahistidine-tagged proteins were cloned into a modified pET28a vector containing an N-terminal hexahistidine tag followed by a protease cleavage site (PreScission) by using NdeI and NotI restriction sites (52). Glutathione S-transferase (GST)–tagged proteins were cloned into a pGEX-6P1 vector by using the BamHI and NotI restriction sites. Mutants were generated by QuikChange mutagenesis and confirmed by DNA sequencing. Details of bacterial expression constructs are shown in table S1.

Protein expression and purification

Proteins were expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells (Stratagene) in Luria-Bertani media and induced at an OD 600 (optical density at 600 nm) of 0.8 with 0.5 mM isopropyl-β-d-thiogalactopyranoside. Details regarding expression times and temperatures are given in table S1. Seleno-L-methionine (SeMet)–labeled proteins were expressed with a methionine pathway inhibition protocol, as previously described (53). Cells were harvested by centrifugation and resuspended in a buffer containing 20 mM TRIS [tris(hydroxymethyl)aminomethane, pH 8.0], 500 mM sodium chloride, and 5 mM 2-mercaptoethanol (β-ME) for hexahistidine-tagged proteins, or a buffer containing 20 mM TRIS (pH 8.0), 200 mM sodium chloride, and 4 mM DTT for GST-tagged proteins; both were supplemented with complete EDTA-free protease inhibitor cocktail (Roche), unless otherwise noted. For purification, the cell suspensions of all proteins and protein complexes were supplemented with 1 mg deoxyribonuclease I (Roche) and lysed by using a cell disruptor (Avestin). Cell lysates were cleared by means of centrifugation at 30,000g for 1 hour. The supernatants were filtered through a 0.45-µm filter (Millipore) and purified using standard chromatography methods (details are given in table S2). Proteins were concentrated to ~10 to 20 mg/ml for biochemical interaction experiments, complex reconstitution, and crystallization. Representative chromatograms and SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analyses are shown for the purifications for Nup120•Nup37•ELYS•Nup85, Sec13•Nup145C, Nup84•Nup133, Nic96•CNT, Nup192, Nup188, Nup170, Nup53, and Nup145N (figs. S51 to S59).

Reconstitution of Nup120•Nup37•ELYS•Nup85

Purified Nup120•Nup37•ELYS heterotrimer was mixed with a twofold molar excess of purified Nup85, incubated for 30 min on ice, and injected onto a HiLoad Superdex 200 16/60 prep grade (PG) gel filtration column equilibrated in 20 mM TRIS (pH 8.0), 150 mM sodium chloride, 5 mM dithiothreitol (DTT), and 5% (v/v) glycerol. Complex-containing fractions were pooled and concentrated to ~8 mg/ml for biochemical studies and further reconstitutions (see also fig. S51 for alternative copurification).

Reconstitution of Nup120•Nup37•ELYS•Nup85•Sec13•Nup145C (CNC hexamer)

Purified Nup120•Nup37•ELYS•Nup85 heterotetramer was mixed with a twofold molar excess of purified Sec13•Nup145C heterodimer, incubated for 30 min on ice, and injected onto a HiLoad Superdex 200 16/60 PG gel filtration column equilibrated in 20 mM TRIS (pH 8.0), 150 mM sodium chloride, 5 mM DTT, and 5 % (v/v) glycerol. Complex-containing fractions were pooled and concentrated to ~8 mg/ml for biochemical studies and further reconstitutions.

Reconstitution of Nup120•Nup37•ELYS•Nup85•Sec13•Nup145C•Nup84•Nup133∆NTE (CNC octamer)

Purified Nup120•Nup37•ELYS•Nup85•Sec13•Nup145C (CNC hexamer) was mixed with a 1.1-fold molar excess of purified Nup84•Nup133ΔNTE heterodimer, incubated for 30 min on ice, and injected onto a HiLoad Superdex 200 16/60 PG gel filtration column equilibrated in 20 mM TRIS (pH 8.0), 150 mM sodium chloride, 5 mM DTT, and 5 % (v/v) glycerol. Complex-containing fractions were pooled and concentrated to ~8 mg/ml for biochemical studies and further reconstitutions.

Reconstitution of Nic96SOL•SUMO-Nup531–90

Purified Nic96SOL was mixed with a 1.2-fold molar excess of purified SUMO-Nup531–90, incubated on ice for 30 min, and injected over a HiLoad Superdex 200 16/60 PG column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 100 mM sodium chloride, and 5 mM DTT. Complex-containing fractions were pooled and concentrated at room temperature to ~10 mg/ml for biochemical studies.

Purification of Nic96•CNT

Cells containing Nic96 and CNT were grown individually and resuspended in 50 mM TRIS (pH 8.0), 500 mM sodium chloride, 4 mM β-ME, and 10 % (v/v) glycerol supplemented with complete EDTA-free protease inhibitor cocktail, 2 mM bovine lung aprotinin (Sigma), and 1 mM phenylmethanesulfonyl fluoride (Sigma). Before lysis, cell suspensions containing CNT and Nic96 were mixed in a 3:1 ratio. Clarified lysate was loaded onto a Ni–nitrilotriacetic acid column equilibrated in a buffer containing 25 mM TRIS (pH 8.0), 500 mM sodium chloride, 4 mM β-ME, 20 mM imidazole, and 5 % (v/v) glycerol and eluted with an imidazole gradient. Peak fractions were pooled and desalted using a HiPrep 26/20 Desalting column (GE Healthcare) equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 150 mM sodium chloride, 5 mM DTT, and 5 % (v/v) glycerol in the presence of ULP1 protease. After SUMO cleavage, the protein was loaded onto a MonoQ 5/50 GL column (GE Healthcare) equilibrated in 20 mM TRIS (pH 8.0), 150 mM sodium chloride, 5 mM DTT, and 5 % (v/v) glycerol and eluted with a sodium chloride gradient. Complex-containing fractions were pooled for further biochemical reconstitutions (see also fig. S54).

Reconstitution of Nup192•Nup145N, Nup192•Nup53, and Nup192•Nup145N•Nup53

Purified Nup192 was mixed with a 1.5-fold molar excess of purified Nup53 and/or Nup145N. The mixture was incubated on ice for 30 min before injection onto a HiLoad Superdex 200 16/60 PG column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 100 mM sodium chloride, and 5 mM DTT. Complex-containing fractions were pooled and concentrated to ~10 mg/ml for biochemical studies and further reconstitutions.

Reconstitution of Nup192•Nic96•Nup53•CNT and Nup192•Nic96•Nup145N•Nup53•CNT (IRC•Nup53)

Purified Nup192•Nup53 heterodimer or Nup192•Nup145N•Nup53 heterotrimer was mixed with a twofold molar excess of purified Nic96•CNT heterotetramer, incubated for 30 min on ice, and injected onto a HiLoad Superdex 200 16/60 PG gel filtration column equilibrated in 20 mM TRIS (pH 8.0), 100 mM sodium chloride, 5 mM DTT, and 2% (v/v) glycerol. Complex-containing fractions were pooled and concentrated to ~5 mg/ml for biochemical studies.

Reconstitution of Nup82NTD•Nup159T•Nup145N

Purified Nup82NTD•Nup159T heterodimer (T, TAIL; residues 1440 to 1481) was mixed with a 1.2-fold molar excess of purified Nup145N, incubated on ice for 30 min, and injected onto a HiLoad Superdex 200 16/60 PG column equilibrated in buffer containing 20 mM TRIS (pH 8.0), 100 mM sodium chloride, and 5 mM DTT. Complex-containing fractions were pooled and concentrated to ~30 mg/ml for biochemical studies.

Reconstitution of Nup170•Nup53, Nup170•Nup145N, and Nup170•Nup53•Nup145N

Purified Nup170 was mixed with a twofold molar excess of purified Nup53 and/or Nup145N. The mixture was incubated on ice for 30 minutes before injection onto a HiLoad Superdex 200 16/60 PG column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 200 mM sodium chloride, 5 mM DTT, and 5% (v/v) glycerol. Complex-containing fractions were pooled and concentrated to ~10 mg/ml for biochemical studies and further reconstitutions.

Reconstitution of Nup170•Nup53•Nup192

Purified Nup170•Nup53 heterodimer was mixed with a 1.2-fold molar excess of purified Nup192, incubated on ice for 30 min, and injected onto a HiLoad Superdex 200 16/60 PG column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 150 mM sodium chloride, and 5 mM DTT. Complex-containing fractions were pooled and concentrated to ~8 mg/ml for biochemical studies.

Reconstitution of Nup188•Nup145N

Purified Nup188 was mixed with a twofold molar excess of purified Nup145N, incubated on ice for 30 min, and injected onto a HiLoad Superdex 200 16/60 PG column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 100 mM sodium chloride, and 5 mM DTT. Complex-containing fractions were pooled and concentrated to ~10 mg/ml for biochemical studies and further reconstitutions.

Reconstitution of Nup188•Nic96•Nup53•CNT

Purified Nup188 was mixed with twofold molar excess of purified Nup53 and Nic96•CNT heterotetramer, incubated for 30 min on ice, and injected onto a HiLoad Superdex 200 16/60 PG gel filtration column equilibrated in 20 mM TRIS (pH 8.0), 100 mM sodium chloride, 5 mM DTT, and 2% (v/v) glycerol. Complex-containing fractions were pooled and concentrated to ~5 mg/ml for biochemical studies.

Reconstitution of scNup170CTD•scNup145N and scNup157CTD•scNup145N

Purified scNup170CTD or scNup157CTD was mixed with a 1.5-fold molar excess of purified scNup145N. The mixture was incubated on ice for 30 min prior to injection over a HiLoad Superdex 200 16/60 PG column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 300 mM sodium chloride, 5 mM DTT, and 5% (v/v) glycerol. Complex-containing fractions were pooled and concentrated to ~10 mg/ml for biochemical studies.

Reconstitution of scNup170CTD•scNup100

Purified scNup170CTD was mixed with a 1.5-fold molar excess of purified scNup100, incubated on ice for 30 min, and injected onto a HiLoad Superdex 200 16/60 PG column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 100 mM sodium chloride, 5% (v/v) glycerol, and 5 mM DTT. Complex-containing fractions were pooled and concentrated to ~10 mg/ml for biochemical studies.

Multiangle light scattering coupled to analytical size-exclusion chromatography

Purified proteins and complex formations were characterized by inline multiangle light scattering (MALS) after separation on a Superdex 200 10/300 GL or a Superose 6 10/300 GL column. All experiments conducted using the Superdex 200 10/300 GL column were performed in a buffer containing 20 mM TRIS (pH 8.0), 100 mM sodium chloride, and 5 mM DTT, whereas experiments conducted using the Superose 6 10/300 GL column were performed in a buffer containing 20 mM TRIS (pH 8.0), 150 mM sodium chloride, and 5 mM DTT. The chromatography system was connected in series with an 18-angle light-scattering detector (DAWN HELEOS II, Wyatt Technology), a dynamic light-scattering detector (DynaPro Nanostar, Wyatt Technology), and a refractive index detector (Optilab t-rEX, Wyatt Technology). Data were collected at 21°C every 1 s at a flow rate of 0.4 ml/min and analyzed using ASTRA 6 software, thereby generating molar mass and mass distribution (polydispersity) measurements of the samples (54). For interaction studies, proteins were mixed and preincubated on ice for 30 min before being applied to the gel filtration column. Protein-containing fractions were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. Experimental and theoretical masses of all proteins and complexes are listed in table S10.

Immunoblotting

Protein from peak fractions was resolved on a 12% SDS-PAGE gel, transferred to polyvinylidene difluoride membranes, and immunoblotted with a mouse antibody against the aviTag (Genscript, 1:1000 dilution), followed by an alkaline phosphatase-conjugated goat antibody against mouse immunoglobulin G (Promega, 1:4000 dilution). Blots were developed with SigmaFast bromochloroindolyl phosphate–nitro blue tetrazolium tablets (Sigma) for 2 to 3 min and imaged immediately.

Crystallization and structure determination

Crystallization trials for all proteins were performed at 21°C in hanging drops containing 1 µl of protein and 1 µl of reservoir solution. SeMet-labeled crystals were grown under similar conditions. Details of the crystallization and cryoprotection are given in table S4. X-ray diffraction data were collected at 100 K at beamline 8.2.2 at the Advanced Light Source (ALS) at Lawrence Berkley National Laboratory, beamline BL12-2 at the Stanford Synchrotron Radiation Source (SSRL), and beamline GM/CA-CAT 23ID-D at the Advanced Photon Source (APS). The x-ray diffraction data were processed using XDS (55). Structures were phased with Phaser, and maps were improved by density modification with RESOLVE (56, 57). Iterative rounds of model building and refinement were performed using COOT and PHENIX (58, 59). All models had excellent stereochemistry and geometry, as determined by Molprobity (60). Details of refinement and data processing are given in tables S5 to S9.

Structure determination of Nup170NTD•Nup53329–361

Purified Nup170NTD was mixed with a threefold molar excess of synthesized Nup53329–361 peptide (Zhejiang Ontores Biotechnologies). To obtain high-resolution diffraction, residues 293 to 305 of Nup170NTD, which were predicted to reside in a large loop, were deleted. Crystals were grown in 1.0 M sodium potassium phosphate (pH 6.9) and cryoprotected by gradual addition of glycerol to the crystallization drop. The Nup53329–361 peptide was added to all cryoprotectant solutions. The structure was determined by single-wavelength anomalous dispersion (SAD) with Phaser, using anomalous x-ray diffraction data collected from a crystal containing SeMet-labeled Nup170NTD.

Structure determination of apo Nup170CTD

A quadruple methionine mutant (Y905M, L1007M, L1183M, and V1292M) was used for crystallization and collection of SeMet SAD data for apo Nup170CTD. Crystals were grown in 0.1 M MES (pH 6.3), 10% (w/v) PEG-20,000 (polyethylene glycol, molecular weight 20,000), 10% (v/v) ethylene glycol, and 0.2 M potassium thiocyanate and cryoprotected by gradual addition of ethylene glycol to the crystallization drop. The four additional methionine positions were introduced as sequence markers for high-confidence sequence assignment. An initial model was used for molecular replacement in Phaser with diffraction data collected from crystals of native apo Nup170CTD that diffracted to 2.1 Å resolution. Crystals of native apo Nup170CTD were grown in 0.1 M Hepes (pH 7.0) and 1.0 M sodium acetate and cryoprotected by gradual addition of ethylene glycol to the crystallization drop.

Structure determination of Nup170CTD•Nup145N729–750

Purified Nup170CTD was incubated with a threefold molar excess of synthesized Nup145N729–750 peptide (Zhejiang Ontores Biotechnologies). Attempts to obtain crystals with stoichiometric Nup145N incorporation revealed that two regions of Nup170CTD could bind to the Nup145N-binding pocket (1403 to 1416 and 1375 to 1377), blocking the Nup170-Nup145N interaction. To obtain crystals of the complex, both of these regions of Nup170 were deleted. Complex crystals were grown in 0.2 M lithium acetate and 12% (w/v) PEG-3350. Crystals were cryoprotected by gradual addition of ethylene glycol to the crystallization drop. Phasing was performed by means of molecular replacement in Phaser, using the structure of apo Nup170CTD as a search model.

Structure determination of Nup170SOL

Crystals of Nup170SOL were grown in 0.1 M Hepes (pH 7.1) and 0.5 M ammonium sulfate and cryoprotected by gradual addition of ethylene glycol to the crystallization drop. The Nup170SOL structure was determined with the MR-SAD routine in PHENIX, combining molecular replacement phases obtained by using the corresponding fragments of the Nup170NTD and Nup170CTD structures with SAD phases obtained from anomalous x-ray diffraction data that were collected from a crystal containing SeMet-labeled Nup170SOL. The crystals diffracted with a high degree of anisotropy, with diffraction past 3.9 Å for two axes, but diffraction was limited to 4.5 Å for the third axis. Minimal model building was performed after the high-resolution structures were refined as rigid bodies, with the exception of the loops connecting helices α16, α17, and α18, which were not observed in the other structures. The overall correctness of the observed conformation was confirmed by calculating anomalous difference Fourier maps to locate the methionine positions. Refinement was performed with anisotropically truncated data, which yielded more interpretable maps.

Structure determination of apo Nic96SOL and Nic96SOL•Nup5331–84

The structure of apo Nic96SOL was determined using SAD x-ray diffraction data collected from crystals grown with SeMet-labeled Nic96SOL in 4% (v/v) tacscimate (pH 7.4) and 14% (w/v) PEG-3350 and cryoprotected by exchanging the crystallization buffer with paratone-N. Purified Nic96SOL•Nup5331–84 was crystallized in 0.1 M TRIS (pH 7.0) and 12% (w/v) PEG-6000, and 1.2 M sodium chloride, and crystals were also cryoprotected by exchanging the crystallization buffer with paratone-N. An initial model of apo Nic96SOL was built using the experimental electron density map and used as a search model in molecular replacement to determine the structure of Nic96SOL•Nup5331–84 at 2.65 Å resolution. Residues 31 to 66 of Nup53 are presumed to be disordered, because only residues 67 to 84 were visible in the electron density map.

Structure determination of Nup192∆HEAD

Despite extensive optimization and screening, crystals of Nup192ΔHEAD diffracted to ~10 Å resolution at best. Inspection of the crystal structure of Nup192NTD suggested helix α9, which protrudes from the surface of the protein, might be preventing the formation of stable crystal contacts. Thus, we replaced residues 167 to 184 with a GSGS linker. Crystals of this mutant grown in 0.1 M TRIS (pH 7.9), 6% (w/v) PEG-4000, and 5% (v/v) polypropylene glycol displayed improved diffraction to ~5 Å resolution. While screening for heavy metal derivatives, we noticed that the diffraction quality of these crystals was improved further by soaking with 0.2-µl saturated potassium hexachloroosmate (K2OsCl6) solution in a total drop volume of 5 µl for 1 min. Crystals were cryoprotected by gradually supplementing the drop with polypropylene glycol in 5% steps. The structure was determined by molecular replacement with Phaser, using the corresponding fragments of Nup192NTD [Protein Data Bank identifier (PDB ID) 4KNH] and Nup192TAIL (PDB ID 5CWV), followed by SAD phasing, using anomalous x-ray diffraction data collected at the osmium L3 edge (15, 20). Although the crystals only grew in the presence of Nup5331–67 peptide, no strong electron density was observed for the peptide, probably because of partial occupancy of the peptide in the crystal.

Structure determination of Nup53RRM

Nup53RRM crystallized in two different crystal forms. Crystals grown in 0.1 M Hepes (pH 7.0), 24% (w/v) PEG-3350, and 0.2 M potassium iodide crystallized in the space group P212121. Crystals were cryoprotected by gradual addition of ethylene glycol to the crystallization drop. The crystal structure was determined by SAD phasing with Phaser, using anomalous x-ray diffraction data from a crystal grown with SeMet-labeled Nup53RRM. Iodide ions were placed in the native structure based on peaks in the anomalous difference Fourier map. These crystals diffracted exceptionally well, with diffraction clearly visible up to 0.7 Å resolution, but the geometry of the beamline did not permit positioning of the detector close enough to measure all spots with very high Bragg angles. As a result, the completeness of the data in the highest-resolution shells is low, but these data were included because they improved the quality of the electron density maps. The second crystal form of Nup53RRM crystallized in the space group P3121 in 4% (v/v) tacsimate (pH 4.1) and 15% (w/v) PEG-3350. The crystals were cryoprotected in 32% (v/v) tacsimate (pH 4.1) and 19% (w/v) PEG-3350. The structure was determined by SAD phasing with Phaser, using anomalous x-ray diffraction data collected from potassium hexachloroosmate–derivatized native crystals.

Structure determination of Nup145NAPD•Nup145CN

Crystals of Nup145NAPD were grown in 0.1 M citric acid (pH 3.0) and 25% (w/v) PEG-3350 and cryoprotected with mother liquor supplemented with 25% (v/v) ethylene glycol. The structure was determined by molecular replacement with Phaser, using the crystal structure of Nup145NAPD (PDB ID 5CWW) as a search model (15). We also crystallized a catalytically dead mutant, Nup145NAPD T994A, that was fused to the N-terminal six residues of Nup145C. Crystals grew in 0.1 M TRIS (pH 8.8), 32% (w/v) PEG-4000, and 0.2 M lithium sulfate and were cryoprotected with mother liquor supplemented with 10% (v/v) glycerol. The structure was determined by molecular replacement with Phaser, using the crystal structure of Nup145NAPD as the search model.

Generation of full-length structures by superposition for docking into cryo-ET reconstructions

Superposition-generated structures of full-length Nup170 and Nup192 were generated by careful superposition of crystal structures of the respective fragments. For Nup170, the crystal structure Nup170NTD•Nup53329–361 was superposed onto the crystal structure of Nup170SOL. Minor differences in the conformations of helices were observed at the truncated ends of the structures, but the conformation included in the models always corresponded to the structure in which no truncation was made. The various structures of Nup170CTD were superposed onto the crystal structure of Nup170SOL by using only helices α19 to α24, which displayed only minimal conformation rearrangements. In the superposition-generated full-length Nup170 structure, residues 1 to 808 were derived from the Nup170NTD•Nup53329–361 crystal structure, residues 809 to 844 were derived from the Nup170SOL crystal structure, and residues 845 to 1416 were derived from the various Nup170CTD crystal structures (see also fig. S25 and Movie 1). A similar approach was used to superpose the structure of Nup192NTD (PDB ID 4KNH) and Nup192TAIL (PDB ID 5CWV) onto the crystal structure of Nup192ΔHEAD, superposing only helices that had identical conformations in both crystal structures (15, 20). In the full-length Nup192 superposition-generated structure, residues 1 to 253 were derived from the Nup192NTD crystal structure, residues 254 to 1750 from the Nup192ΔHEAD crystal structure, and residues 1750 to 1756 from the Nup192TAIL crystal structure (see also fig. S31 and Movie 4).

Docking crystal structures into the cryo-ET reconstruction of the intact human NPC

An incremental approach was used to dock available crystal structures and superposition-generated structures into the previously reported ~23 Å cryo-ET reconstruction of the intact human NPC (28). For all structures other than Nup37, Nup43, and Nup133NTD, global searches in the cryo-ET reconstruction were performed using the UCSF Chimera Fit Map command (61). Searches consisted of 50,000 initial placements that were locally optimized and scored based on correlation between a simulated 20 Å map of the docked model and the cryo-ET reconstruction, taking into account the C8 rotational symmetry of the latter. The top-scoring solutions for the search were inspected for their agreement with previously published biochemical data and the biochemical data presented here. A flowchart of our incremental docking approach can be found in fig. S34. The average Fourier shell correlation between a single spoke and the map corresponding to the docked spoke was calculated using Refmac5 to a value of 0.79 at the reported resolution of the map (23 Å) (62).

Docking of the CNC

Searches for the CNC hexamer (PDB ID 4XMM) and the hsNup84•hsNup133 heterodimer (PDB ID 3CQC) were performed in maps from which the density corresponding to the nuclear envelope had been removed (14, 38). Because of its large size and distinctive shape, the four top-scoring solutions of the CNC hexamer have much higher scores than any other solution. Nup37 was docked by superposing the crystal structure of the S. pombe Nup120NTD•Nup37 heterodimer (PDB ID 4FHN) onto the CNC hexamer (39). hsNup43 was docked into an unoccupied density next to Nup85, based on a previously identified cross-link and the previously reported interaction (13, 41). hsNup133 (PDB ID 1XKS) was docked in an unoccupied density next to the Nup120 β-propeller, and the fit was optimized locally with the Fit Map command (63). Because the resolution of the map cannot distinguish between different orientations of the Nup43 and Nup133 β-propellers, their exact orientation is ambiguous. Details regarding CNC docking can be found in fig. S35.

Docking of Nup170, Nup192, and Nup188

Before performing global searches for Nup170 and Nup192, the density corresponding to the docked CNCs was removed to obtain placements that would not clash with the CNCs. Searches in maps that included the density assigned to the CNCs produce similar results. Searches with Nup170 were performed using multiple conformations. Most conformations produced similar results, referred to as conformation I, and occupied two symmetry-related positions in the inner ring. A different conformation (conformation II), which was derived from the structure of the apo Nup170CTD quadruple methionine mutant, occupied symmetry-related positions bridging the inner and outer rings. For Nup192, we accepted five placements from the global search results, two placements in the inner ring, two placements in the outer rings, and one placement on the nuclear peripheral side of the inner ring. A matching cytoplasmic peripheral placement was not found in global searches, but the manual placement on the cytoplasmic peripheral side of the inner ring generated a score that would have ranked as the 9th highest. The four molecules placed in the inner ring were assigned as Nup192, and the two molecules placed in the outer rings were assigned as Nup188, based on the following considerations: (i) Nup188 binding to Nup145N was incompatible with both Nup192 and Nup170, making it unlikely that Nup188 is a component of the tightly packed inner ring core; (ii) cross-linking and mass spectrometry data identified cross-links between Nup188 and Nup85 (13, 42); and (iii) Nup188 mislocalization behavior was found to be consistent with other outer ring components upon genetic perturbation (64). Details regarding Nup170, Nup192, and Nup188 docking can be found in fig. S36.

Docking of Nic96SOL•Nup5331–84 and CNT•Nic96R1

Searches for the Nic96SOL•Nup5331–84 complex were performed using a map of the inner ring from which the density corresponding to the placed Nup170, Nup188, and Nup192 molecules had also been removed. Many high-scoring solutions of Nic96SOL were related to each other by rotations along the long axis of the rod-shaped molecule, reflecting the ambiguity of the exact rotational orientation of the molecule at the resolution of the cryo-ET reconstruction. Searches for the CNT•Nic96R1 complex (PDB ID 5CWS) were performed in a map from which the density corresponding to the placed Nic96SOL molecules also had been removed (15). Superposition of the X. laevis (xl) CNT crystal structures (PDB IDs 5C2U and 5C3L) was performed by superposing xlNup54 from the xlCNT structure directly onto Nup57 from the CNT structure, and then superposing the ferredoxin-like domain onto the xlCNT structure (16). Details regarding Nic96SOL•Nup5331–84 and CNT•Nic96R1docking can be found in fig. S37.

Manual identification of a third pair of Nup170 molecules

After successful placement of 32 copies each of Nup170, Nup192, Nic96SOL, and the CNT in the inner ring, we continued our analysis by removing the density corresponding to the docked molecules. There were four major distinct densities remaining in the inner ring, each of which appeared on both the nuclear and cytoplasmic sides (fig. S40). The first set of densities corresponded to the ferredoxin-like domain that is only present in metazoan Nup57. The second set corresponded to the density adjacent to the C-terminal TAIL domains of the equatorial Nup192 molecules, which we assume arises from our structure imperfectly capturing the conformation of Nup192 in the assembled NPC. The third major density was adjacent to the equatorial Nup170 β-propellers. We were unable to assign this density confidently to any remaining subunit, such as Nup53RRM, Nup145NAPD, or any other nucleoporin of known structure. Lastly, a pair of extended densities was adjacent to the nuclear envelope (fig. S40). These densities were very similar in shape to the Nup170 molecules that we had already placed, but neither of the conformations that we previously placed yielded a high-quality fit. Instead, we found the best fit with a third conformation, although visible differences suggest that even this structure did not perfectly capture the conformation of this third molecule inside the NPC. Details regarding docking for this conformation of Nup170 can be found in fig. S40.

Protein labeling for oligomerization experiments

Proteins were labeled under conditions that were selective for the N-terminal amino group of proteins. Before the labeling reaction, purified CNC hexamer, Nup84•Nup133, and Nup84•Nup133ΔNTE complexes at concentrations of 10 to 20 µM were dialyzed in a buffer containing 100 mM sodium bicarbonate (pH 8.0), 100 mM sodium chloride, and 5 mM DTT. 20 µM Nup170 and Nup170ΔC were dialyzed against a buffer containing 100 mM sodium bicarbonate (pH 8.0), 200 mM sodium chloride, and 5 mM DTT. For the labeling reactions, BODIPY (boron-dipyrromethene) FL NHS Ester (succinimidyl ester) or Alexa Fluor 647 NHS Ester (succinimidyl ester) dyes (Molecular Probes) were added at ratios equimolar to the dialyzed protein samples and incubated for 50 min at room temperature. The reaction was subsequently quenched by dialysis against buffers containing 100 mM TRIS (pH 8.0) instead of sodium bicarbonate.

Oligomerization experiments

Oil droplet formation was analyzed by fluorescence microscopy with a Carl Zeiss AxioImagerZ.1 equipped with an AxioCamMRm camera. Experiments performed without Nup170 were performed with BODIPY-labeled CNC hexamer and Alexa Fluor 647–labeled Nup84•Nup133 or Nup84•Nup133ΔNTE. Proteins were mixed at equimolar ratios directly on a cover slide and analyzed immediately. For experiments involving the incorporation of Nup170, BODIPY-labeled CNC hexamer and unlabeled Nup84•Nup133 were first mixed together in equimolar ratios and then subsequently incubated with a 1.5-fold molar excess of Alexa Fluor 647–labeled Nup170 or Nup170ΔC.

Figures and movies

Gel filtration profiles and MALS graphs were generated in IGOR (WaveMetrics) and assembled in Adobe Illustrator. Sequence alignments were generated using MUSCLE and colored with ALSCRIPT (65, 66). Electrostatic potentials were calculated with APBS (Adaptive Poisson-Boltzmann Solver) software (67). All structural figures and movies were generated using PyMol (www.pymol.org).

Supplementary Materials

References and Notes

Acknowledgments: We thank J.-Y. Mock, S. Petrovic, and A. Patke for critical reading of the manuscript. We thank M. Beck for sharing the cryo-ET reconstruction of the intact human NPC before publication; E. Hurt for providing material and discussions; and C. Bayes, S. Butkovich, C. Frick, A. Koehl, H. Kuberczyk, R. Tran, and S. Zimmerman for experimental support. We also acknowledge J. Kaiser and the scientific staff of the SSRL beamline 12-2, the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) at the APS, and the ALS beamline 8.2.1 for their support with x-ray diffraction measurements. We acknowledge the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program for their support of the Molecular Observatory at the California Institute of Technology (Caltech). The operations at the SSRL, ALS, and APS are supported by the U.S. Department of Energy and the National Institutes of Health (NIH). GM/CA has been funded in whole or in part with federal funds from the National Cancer Institute (grant ACB-12002) and the National Institute of General Medical Sciences (grant AGM-12006). D.H.L and A.M.D. were supported by a NIH Research Service Award (5 T32 GM07616). D.H.L. was also supported by an Amgen Graduate Fellowship through the Caltech-Amgen Research Collaboration. T.S. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. F.M.H. was supported by a doctoral fellowship from the Boehringer Ingelheim Fonds. Y.F. was supported by a visiting doctoral student scholarship from the China Scholarship Council. A.H. was supported by Caltech startup funds, the Albert Wyrick V Scholar Award from the V Foundation for Cancer Research, the 54th Mallinckrodt Scholar Award from the Edward Mallinckrodt Jr. Foundation, a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research, a Camille-Dreyfus Teacher Scholar Award, and NIH grant R01-GM111461, and he is an inaugural Heritage Principal Investigator of the Heritage Medical Research Institute. The coordinates and structure factors have been deposited in the PDB with accession numbers 5HAX (Nup170NTD•Nup53R3), 5HAY (Nup170CTD), 5HAZ (Nup170CTD), 5HB0 (Nup170CTD•Nup145NR3), 5HB1 (Nup170SOL), 5HB2 (Nic96SOL), 5HB3 (Nic96SOL•Nup53R2), 5HB4 (Nup192∆HEAD), 5HB5 (Nup145NAPD), 5HB6 (Nup145NAPD•Nup145CN), 5HB7 (Nup53RRM, space group P212121), and 5HB8 (Nup53RRM, space group P3121). A PyMol session file containing the composite structure of the NPC symmetric core can be obtained from our webpage (http://ahweb.caltech.edu). The authors declare no financial conflicts of interest. This work is dedicated to the memory of Mandy Hoelz.
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