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. 2026 Feb 18;12(8):eadz6794. doi: 10.1126/sciadv.adz6794

The vault particle is enclosed by a C13-symmetric cap with a positively charged exterior

Huan Li 1,2,3, Francesca Vallese 4, Oliver B Clarke 1,2,3,*
PMCID: PMC12915596  PMID: 41706843

Abstract

Vaults are some of the largest ribonucleoprotein complexes known and are highly conserved across eukaryotes, but both their function and key details of their architecture remain unclear. While high-resolution structures of the vault shell are available, the architecture and symmetry of the cap have remained unresolved. Here, we present a 2.25-angstrom cryo–electron microscopy structure of the vault cap, revealing an unexpected 13-fold symmetric arrangement that contrasts with the 39-fold symmetry of the vault body, with each repeating module of the cap formed by an asymmetric homotrimer of adjacent subunits. The center of the cap features an unusual architecture, consisting of two concentric β barrels surrounded by an interwoven two-layer stack of α helices. The vault cap features a positively charged exterior and a negatively charged interior surface, with implications for binding partner recruitment and engineering of modified vault particles.

Cryo-EM analysis of the cap of the vault particle reveals an unexpected C13-symmetric architecture.

INTRODUCTION

Vaults are large ribonucleoprotein complexes that are ubiquitously present in eukaryotic cells. The vault was first found in 1986, with an ovoid, highly regular architecture characterized by negative stain electron microscopy (1). The first vault structure was resolved by cryo–electron microscopy (cryo-EM) in 1999, revealing a symmetrical, barrel-like architecture (2). A higher resolution crystal structure conclusively resolved the overall symmetry of the vault shell, showing that it is formed by 78 copies of major vault protein (MVP) arranged with D39 symmetry, but the cap regions at either end of the vault were not resolved, likely due to a symmetry mismatch with the rest of the vault shell (3). In addition to MVP (4), the vault complex includes vault poly(adenosine 5′-diphosphate–ribose)–polymerase (vPARP) (5), telomerase-associated protein 1 (TEP1) (6), and untranslated vault RNAs (vRNAs) (7, 8).

Further insights into vault organization were gained through cryo–electron tomography (cryo-ET), which allowed direct visualization of vaults in intact cells. These studies showed that cargo within the vault lumen is not arranged in an ordered manner with respect to the vault walls, potentially explaining challenges in computational classification of cargo in single-particle cryo-EM (9). In addition, structural analysis of recombinant vaults by cryo-EM demonstrated that vault opening occurs via an expansion at the waist, which may serve as a mechanism for cargo release and uptake (10).

Evolutionary analysis indicates that vaults are highly conserved across multicellular eukaryotes, suggesting an ancient and fundamental cellular role (11), but a detailed understanding of this role remains elusive, although recent studies have suggested a role for vault in both the adaptive and innate immune response. Mvp knockout mice exhibited significantly attenuated immunity responses and decreased survival following homologous influenza A virus rechallenge, in comparison to wild-type mice, suggesting that vaults can enhance adaptive immunity against viral pathogens, either directly or through interactions with innate immune pathways (12). Furthermore, vaults contribute to host resistance against lung infections caused by Pseudomonas aeruginosa (13).

In addition to their role in immune responses, vaults are involved in intracellular transport, particularly through interactions with the microtubule network. They have been shown to move along microtubules (14), and their mobility is, at least in part, dependent on microtubule integrity (15). Moreover, vaults associate with lipid rafts, suggesting a potential role in membrane-related cellular processes (16). One of the most well-characterized functions of vaults is their involvement in drug resistance in cancer. Vaults have been identified as the human lung resistance–related protein, which contributes to chemoresistance through mechanisms that are still under investigation (17). A recent study has shown that vaults may mitigate bone loss associated with aging and estrogen deficiency by promoting Fas-mediated apoptosis in osteoclasts. MVP prevents ubiquitination of target proteins, presumably by sequestering them from the cytosol, and MVP deficiency results in osteoporotic phenotypes in vivo and in vitro (18). The vault also plays crucial roles in the nervous system. vRNAs directly bind mitogen-activated protein kinase kinase 1, amplifying the extracellular signal–regulated kinase signaling pathway, which is essential for synaptic connectivity and plasticity (19). In addition, vaults may be involved in nucleocytoplasmic transport, as evidence suggests that they participate in the exchange of macromolecules between the nucleus and cytoplasm (20). Vaults have been exploited for biotechnological applications, particularly in drug delivery, owing to their large internal cavity, biocompatibility, and ability to be engineered for targeted transport. Recombinant vaults can be functionalized with targeting peptides or loaded with therapeutic molecules, enabling the delivery of drugs, nucleic acids, or proteins to specific cells or tissues with minimal immunogenicity and toxicity (21–23).

Despite these advances, many aspects of vault biology remain unclear. Further research is needed to elucidate their precise mechanisms of action, cargo specificity, and potential biomedical applications. Understanding these fundamental aspects will be crucial for harnessing vaults in therapeutic and biotechnological contexts. Here, using single-particle cryo-EM, we show that the cap of the vault, rather than being open, as might be expected from prior structural studies, is actually a closed and tightly folded assembly, with 13-fold symmetry that contrasts with the 39-fold symmetry of the main body of the vault, and a 15-Å passage through the center of the cap that connects the vault lumen to the cytosol. These results have implications for our understanding of vault architecture and for design and engineering of vaults as delivery vehicles for therapeutics.

RESULTS

High-resolution structure determination of the rabbit vault particle

We purified vault from rabbit liver using two successive density gradient centrifugation steps following previously published protocols (2). This initial preparation yields intact vaults containing MVP, TEP1, and vPARP (fig. S1, A and B).

Cryo-EM analysis of the vault particles on graphene-oxide grids revealed barrel-like particles with the expected D39 apparent symmetry, yielding an initial reconstruction at 2.3 Å with D39 symmetry applied after incorporating Ewald sphere corrections to account for the defocus gradient across the particle. Overall, the vault shell exhibited similar features to previously published vault structures. The vault is 700 Å long and ~400 Å wide (Fig. 1A). Despite extensive classification attempts, we were unable to identify ordered cargo within the vault lumen, consistent with prior cryo-ET results of native vaults on human umbilical vein endothelial cells (9). As for previous vault structures, the density for the cap region was uninterpretable, prompting us to initiate local processing of this region, as outlined below.

Fig. 1. Cryo-EM structure of vault and symmetry determination of vault cap.

Fig. 1.

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(A) The overall map and structure of vault in top and side view. (B) The top and side view of vault cap at C39 symmetry. (C) The top and side view of vault cap in C13 symmetry; red, yellow, and teal represent VA, VB, and VC chain, respectively. (D) Comparison of the conformation and secondary structure of VA, VB, and VC.

To resolve the symmetry of the vault cap, we performed D1 symmetry expansion, and subparticles were extracted centered on the apical entrance of the vault, reconstructed, and refined with C39 symmetry. A block-based reconstruction approach was applied, in which the per-particle defocus values of the subparticles were corrected on the basis of their z-height difference with respect to the center of the vault. In C39, the walls of the vault cap had excellent density, with well-resolved side chains, but the planar cap had indistinct, disordered density that could not be confidently modeled, causing suspicion of a symmetry mismatch (Fig. 1B). Focused classification without alignments in C1, using a mask covering the planar end of the vault, revealed three classes with C13 symmetry, distinguished only by rotation about the pseudo-C39 symmetry axis. Aligning the three volumes, and adjusting the poses of the associated particles by the same transformation, facilitated local refinement with C13 symmetry, revealing the detailed architecture of the vault cap at 2.25-Å resolution (Fig. 1C, fig. S5, and movie S1).

The cryo-EM structure of the vault cap has allowed us to complete the model of the vault shell. Because of the C13 symmetry of the cap, there are three sequence-identical but distinctly folded protomers that comprise the asymmetric unit, which we termed VA, VB, and VC. Each protomer can be divided into 12 domains from the N terminus to the C terminus: nine structural domain repeats [R1 to R9, homologous to SPFH (stomatin, prohibitin, flotillin, and HflK/C) 1 domains], an MVP shoulder domain (SPFH2), a cap helix domain (coiled-coil domain in SPFH nomenclature), and a C-terminal cap disk domain. The cap disk domains include three different distinct folds in the three different protomers (Fig. 1D).

Architecture of the vault cap

The vault shell exhibits almost perfect D39 symmetry until G803. Starting from P804, the 39 chains show three different folds, which weave together to form a two-layer arrangement, in which two layers of α helices orthogonal to the pseudo-C39 axis buttress two concentric β barrels, the outer of which has 26 parallel strands (contributed by VB and VC) and the inner of which has 13 parallel strands (formed solely by VC).

The three protomers have distinct folds, which come together to form the cap disk, as follows (Fig. 2, A to D). In the following description, we numbered the secondary structural elements starting from the first helix after the cap helix domain. The VA chain is mostly α-helical (α1 to α3). VA projects partway toward the center of the cap, and then VA-α2 turns outward, forming a helix-turn-helix. After a missing loop (K828 to V839), α3 stacks together with the α1 helix (Fig. 2B). Thirteen copies of the VA chains form an “outer ring” near the inner wall of the cap (fig. S2A). Second, the VB chain contains helices α1, α2, and a single strand, β1. The curved α1 helix projects all the way to the center of cap, and then the β1 strand forms the first strand in a 26-stranded barrel surrounding the apical entrance of the vault—the “outer barrel”—before emerging in the vault lumen. The luminal VB-α2 helix runs parallel to the cap disk surface (Fig. 2C). Thus, 13 copies of the VB protomer form a “middle ring” around the outline of the inner β barrel (fig. S2B). Last, the VC protomer includes α1, β1, and β2. The VC-α1 helix extends toward the center until L825 running parallel to VB-α1, and then VC-β1 projects into the vault lumen, forming the complementary strand to VB-β1, which together form the outer barrel. VC then makes a 180° turn and forms a putative β strand, β2, which extends out back toward the cytosol and terminates at T844, forming an “inner barrel” (Fig. 2D). Overall, the cap structure looks similar to a sunflower when viewed from the vault lumen (fig. S2C). VA, VB, and VC chains independently form the outer ring, the middle ring, and the inner barrel, respectively, while VB and VC weave together to form the outer barrel.

Fig. 2. Overall architecture of vault cap.

Fig. 2.

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(A) Amino acid sequence of the vault cap. The secondary structures of VA, VB, and VC chains are indicated with cartoons. (B to D) The structures of VA, VB, and VC chains for vault cap. (E) The interactions between VA, VB, and VC chains. (F) The cross section of vault cap. Outer layer represented in magenta, inner layer represented in apricot, outer barrel represented in blue, and inner barrel represented in pink. (G) Beta strand of outer barrel; T830/I832 is adjacent in VB and VC chains. (H) Single VA, VB, and VC chain combination. (I) Inner barrel structure (G835 to A845) overlays with density map; map is contoured at level 0.15. (J) Interior aperture of vault cap. The pore size and hydrophobicity of the barrel were calculated using software CHAP (45). N840 and F842 are labeled, and the side chain of F842 (depicted with transparency) was filled using Dock Prep in ChimeraX before CHAP calculation.

The VC chain interacts with VA chain at the outer ring and with VB chain at the middle ring (Fig. 2E). VC chain acts similar to a bridge, connecting VA and VB chains. L821, L822, and L825 from VC-α1 form a key interprotomer interface. The triple leucine motif from VC interlocks with L841, F842, T844, A845, and F846 from VB-α2. Regarding interactions between VA and VC, I807 in VC-α1 helix interacts with the hydrophobic pocket formed by T806, D809, L810, L821, and L825 from VA-α1 and VA-α2.

The 13 sets of three chains form a two-layer, double-barrel arrangement when looking at the cross section of the vault cap. VB-α1 and VC-α1 form the outer layer. The VA outer ring and VB middle ring form the inner layer. VB and VC chains fold together to form two concentric β barrels in the center of the vault cap, creating a passage to the inside of the vault particle (Fig. 2F). The outer barrel consists of VB-β1 and VC-β1. Adjacent strands in the outer barrel are offset in register by one residue (Fig. 2G). The inner barrel is formed solely by VC-β2. The C termini of both VA and VB chains project into the vault lumen, while the C terminus of VC chain points out into the cytosol (Fig. 2H). This observation has parallels with the heterodimeric arrangement in the HflK/C complex from the SPFH family, in which the HflK subunit points inside while the HflC subunit points out to the extracellular region (24).

The inner barrel forms a dynamic portal between the vault lumen and the cytosol

The inner barrel formed by the VC chain connects the vault lumen and the cytosol. In our dataset, the inner barrel is defined by 13 β strands (Fig. 2I). These strands, while having strong density, are less well resolved than the surrounding regions and lack most side chain densities despite extensive attempts at classification and symmetry relaxed refinement to resolve possible pseudosymmetry. It is possible that the inner barrel is flexible or dynamic, which limits the resolution in this region. Another possibility, which we favor, is that the inner barrel has an additional layer of pseudosymmetry, which is not possible to resolve with the present dataset and currently available computational tools. There is also a strong density in the center of the inner barrel, which could correspond either to one of the termini of the vault protomers or potentially a small molecule or additional protein segment. Although the density for the inner barrel strands is poor, connectivity with the preceding regions of the outer barrel is clear, allowing moderate confidence in sequence assignment for VC-β2. The constriction of the inner barrel pathway is most likely formed by residues N840 and F842, with a diameter of ~15 Å (Fig. 2J). This constriction marks the boundary between the outward-facing hydrophilic region and the inward-facing hydrophobic region of the pathway. Consurf analysis (25) indicates that the inner barrel is highly conserved, especially at N840 (fig. S2F). The 15-Å pore size appears potentially sufficient to allow diffusion of small molecules, ions, and possibly peptides, when the pore is not occluded.

The vault cap forms a planar disk with a positively charged exterior and negatively charged interior

Electrostatic analysis shows that the cap has a positively charged ring formed by K828 from both VB and VC chains on the outer surface (Fig. 3, A and C). This positively charged feature likely mediates interactions with negatively charged partners, such as nucleic acids, anionic lipids, or other proteins. K828 is broadly conserved across vertebrate species, with the exception of Xenopus and chicken, where a glutamine occupies the equivalent position, complemented by positive charges substituted in nearby regions of the cap (Fig. 3B). This substitution maintains the ability for this residue to interact with the negatively charged partners, suggesting that the ring is functionally conserved across species. The cap also has an area of negative charge on the inside (Fig. 3C). The negatively charged interior may form an electrostatic well capable of retaining or repelling small molecules or ions.

Fig. 3. Electrostatics analysis of vault cap.

Fig. 3.

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(A) The structure of the outer barrel. K828 that forms a positive ring is shown in blue. VB chains are shown in teal, and VC chains are shown in yellow. (B) Sequence alignment of MVP from various species, spanning residues K820 to M861 of the rabbit sequence. Residues colored by Consurf analysis. Yellow lane indicates insufficient data. (C) Electrostatic analysis of the vault cap performed using Coulombic surface coloring in ChimeraX, shown from both exterior and interior views. Inset: Zoom-in around the center of the cap from the exterior view. K828 is indicated. (D) Consurf analysis of the central region of the vault cap viewed from the lumen (left) and cytosol (right). K828 is labeled on VB and VC.

DISCUSSION

The vault complex is highly conserved across multicellular eukaryotes. Understanding the fundamental molecular factors that control vault dynamics is crucial not only for unraveling the functions these particles play in various cellular processes but also for enhancing their utility as customized molecular carriers. In this study, we report the high-resolution structure of the vault complex, revealing the architecture and symmetry of the vault cap. To our knowledge, this represents the most detailed structural analysis achieved for the vault complex to date, offering unexpected insights into its organization and potential functions. During preparation of this work, Lövestam and Scheres (26) reported a cryo-EM structure of human vault particles, imaged from brain tissue, which also revealed the presence of C13 symmetry at the caps. Their observations are consistent with our findings and underscore that the C13-symmetric architecture of the vault cap is likely to be a conserved feature across species.

Our data show that the vault cap is formed by 13 copies of an asymmetric homotrimer. While most species encode a single MVP, Dictyostelium discoideum is a notable exception, expressing two isoforms—MVP-A and MVP-B—which share 65.1% sequence similarity. This raises the possibility that D. discoideum vaults may form heterooligomers incorporating both isoforms, which would be particularly interesting given the odd number of MVP copies in a complete vault. On the basis of our results, the MVP shell in D. discoideum could adopt a 2:1 assembly, with alternating A/B/A or B/A/B subunits. Further studies will be needed to determine whether such mixed assemblies occur in vivo.

Previous studies have shown that vRNAs are likely bound near or within the cap and associates with TEP1, but whether the binding site is on the inside or outside has not been conclusively shown (27). Given the positively charged patch on the outside and the negative charge on the inside, it seems likely that vRNAs interact with the positively charged patch on the outside, if they are interacting directly with the vault cap.

The architecture of the vault cap reveals similarities with SPFH family proteins

The cap structure of the vault particle exhibits a distinctive arrangement of two concentric β barrels, reminiscent of architectures observed in members of the protein SPFH superfamily (Fig. 4, A to C). A well-characterized example within this family is the HflK/C complex, whose asymmetric unit forms a heterodimer comprising one HflK and one HflC subunit. These two subunits stack to form a concentric inner barrel with 12-fold (C12) symmetry (Fig. 4B). Specifically, the outer barrel consists of 12 β strands contributed by HflC subunits, while the inner barrel is composed of 12 strands from HflK subunits (24).

Fig. 4. Comparison with SPFH family.

Fig. 4.

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(A) The overall structure of vault shell and zoomed-in structure of cap in three colors. (B) The overall structure of HflK/C complex [Protein Data Bank (PDB): 7WI3] and zoomed-in structure of cap; HflC and HflK in two colors. (C) The overall structure of flotillin complex (PDB: 9BQ2) and zoomed-in structure of cap; Flotillin-1 and Flotillin-2 in two colors. (D) The overall structure of Erlin complex (PDB: 9O9U) and zoomed-in structure of cap; Erlin1 and Erlin2 in two colors. (E) The overall structure of stomatin complex and zoomed-in structure of cap; stomatin A and stomatin B in two colors.

A comparable structural arrangement is observed in the flotillin complex, in which the C-terminal regions of Flotillin-1 and Flotillin-2 assemble into a single β barrel exhibiting C22 symmetry (Fig. 4C). Although the inner β barrel is not explicitly modeled in the resolved structure of the flotillin complex, density map analysis suggests the possible presence of an unmodeled internal barrel (28, 29). Additional members of the SPFH family, including the endoplasmic reticulum–resident Erlin complex (30) and the mitochondrial prohibitin complex (31), have recently been reported to form higher-order assemblies with similar architectural principles. In particular, the C-terminal region of the Erlin complex forms a layered structure that resembles the concentric organization seen in the vault cap, including a 13-stranded inner β barrel at the center of a 26-stranded outer barrel, although with different connectivity to that observed in the inner and outer barrel arrangement in the vault cap.

While the vault cap shares these common architectural motifs with other members of the SPFH family, it also exhibits unique characteristics. Unlike the heteromeric assemblies typical of most other SPFH family complexes, the vault shell is composed of 13 copies of an asymmetric homotrimer. Although the three protomers are chemically identical, they adopt distinct conformations, resulting in a nonequivalent trimeric fold that contributes to the characteristic architecture of cap. One other homooligomeric SPFH family member is stomatin, which is composed of eight copies of an asymmetric homodimer, where an identical sequence adopts two different conformations at the C terminus of stomatin, giving an inner β barrel with 16 strands but C8 symmetry (Fig. 4E) (32, 33). Refinement of stomatin in C16, before resolving the C8 symmetry, resulted in a map with featureless β strands, similar to what we see for the inner barrel of the vault.

Electrostatics of the vault cap have implications for associations with binding partners and cellular compartments

The electrostatic properties of the vault cap are essential for mediating its interactions with various cellular structures and binding partners. Notably, a prominent positively charged region is observed on the cytosolic side of the vault cap. This feature is consistent with previous observations demonstrating an end-on interaction between vault particles and microtubules, suggesting that such associations occur preferentially via the cap rather than the barrel domain of the vault (34). This spatial specificity in microtubule binding underscores the importance of charge distribution in directing vault positioning and trafficking within the cell.

In addition to its interaction with cytoskeletal elements, the vault cap has also been implicated in the binding of vRNAs. It has been proposed that this interaction is mediated by TEP1, which associates with both the vault and vRNAs. However, the exact binding site of vRNAs—whether internal or external to the cap—remains unresolved (27). The distinct electrostatic patterning of the cap offers potential insights into this question. Specifically, electrostatic surface potential mapping reveals a positively charged exterior and a predominantly negatively charged interior within the cap structure. This distribution suggests that if vRNAs interact directly with the vault cap, the interaction is more likely to occur on the outer surface, where electrostatic attraction would be maximized.

Implications for design and engineering of modified vault particles

The structural features of the vault cap provide a framework for the rational design and engineering of modified vault particles. Its defined architecture allows for structure-guided modifications of the vault cap to enhance stability and fine-tune surface properties such as charge and hydrophobicity. Given that vault particles assemble from an asymmetric homotrimer, engineering efforts could focus on generating heterotrimeric or heterodimeric vaults with selectively stabilized conformations for each protomer. One potential approach involves truncating the segment between VA-α2 and VA-α3 to restrict flexibility, thereby favoring the VB chain conformation. In addition, mutating G847 to alanine in VC chain may increase the α-helical propensity of this region, further stabilizing the desired structural arrangement. These modifications could provide a means to control vault assembly and improve functional specificity. Selective stabilization of conformations may enable the precise fusion of cargo within the vault interior while preserving structural integrity. Furthermore, engineered vaults could be designed to display targeting motifs or functional domains on specific protomers that project into the cytosol while carrying cargo in the lumen. These modifications could expand the utility of vaults in targeted drug delivery, molecular transport, or synthetic biology applications. Overall, a structure-informed approach to vault engineering has the potential to enhance their modification for biomedical and nanotechnological applications.

MATERIALS AND METHODS

Purification of vaults

Vaults were purified essentially as previously described in Kong et al. (2). Briefly, Rabbit liver (BioIVT) was homogenized using a polytron in buffer A containing 75 mM NaCl, 50 mM tris-HCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and Pierce protease inhibitor mini tablet [Thermo Fisher Scientific (TFS)], with pH adjusted to 7.4. Debris was removed by centrifugation at 500g for 15 min. The supernatant was passed through cheesecloth and centrifuged at 20,000g for 20 min. The final supernatant was centrifuged again at 100,000g for 2 hours to obtain a crude microsome. The crude microsomes were then resuspended in buffer A containing 6.25% sucrose and 6.25% Ficoll 70 (Sigma Chemical Co.) and centrifuged at 40,000g for 40 min. The supernatant was diluted with 4 volumes of buffer A and pelleted down by 100,000g for 2 hours. The pellet was resuspended in buffer A with CsCl (0.5 g/ml), loaded onto a step gradient of CsCl steps (1.45, 1.50, and 1.70 g/ml), and centrifuged in a SW 32 rotor for 24 hours at 55,000g. The load fraction was saved, diluted with 4 volumes of buffer A, and centrifuged for 3 hours at 100,000g. The pellet was resuspended again in buffer A plus CsCl (0.5 g/ml), loaded onto a second CsCl step gradient, and fractionated as described. Final purified vaults from the load fraction were resuspended in phosphate-buffered saline containing 1 mM MgCl2, 0.5 mM EGTA, and protease inhibitor cocktail.

Negative stain imaging

Negative staining was performed using established methods (35). Briefly, 200-mesh copper grids covered with carbon-coated collodion film (Electron Microscopy Sciences, Hatfield, PA, USA) were glow discharged for 30 s at 10 mA in a PELCO easiGlow glow discharge unit (PELCO, Fresno, CA, USA). A total of 3 μl of purified vault sample (50 μg/ml) was adsorbed to the grids and incubated for 30 s at room temperature. Samples were then washed with two drops of water and stained with two successive drops of 0.3% (w/v) uranyl formate (electron microscopy sciences, Hatfield, PA, USA), followed by blotting until dry. The sample was collected using a Tecnai Spirit T12 transmission electron microscope operated at 120 kV (Thermo Fisher Scientific, Waltham, MA, USA) at a nominal magnification of ×26,000 [2.34 Å per pixel (px)].

Cryo-EM data collection

A total of 3 μl of purified vaults at 2 mg/ml was added to a glow-discharged (PELCO easiGlow) homemade graphene oxide (GO) grid [Quantifoil UltrAuFoil grids (R0.6/1, Au 300-mesh gold)] and blotted for 5 s at 4°C and 100% humidity using the Vitrobot Mark IV system (TFS) before plunging immediately into liquid ethane for vitrification. For homemade GO grids, 2.4% (w/v) GO solution (Sigma-Aldrich) dissolved in 20% ethanol solution was vortexed and centrifuged at 3000 rpm for 5 min. A total of 1 μl of the supernatant was added on the top of each grid and allowed to air dry. Images were acquired on a Titan Krios electron microscope (TFS) equipped with a K3 direct electron detector (Gatan) and a Gatan Imaging Filter operated at ×105,000 magnification with 0.4125 Å per px in super-resolution mode (0.825-Å physical pixel size) using SerialEM automated data collection software (36) . Pixel size is calibrated by cross-correlation with vault crystal structure Protein Data Bank (PDB): 4HL8. Data collection was performed using a dose of 47.86 e−/Å2 across 50 frames (50 ms per frame) at a dose rate of 13.03 e− px−1 s−1 using a set defocus range of −0.5 to −1.5 μm. A 100-μm objective aperture was used. Data were collected using image shift with hardware coma correction. A total of 11,213 micrographs were collected.

Cryo-EM data processing

The brief cryo-EM data processing workflow is summarized in fig. S3. Euler angle distributions, conical Fourier shell correlation plots, and validation statistics are presented in fig. S4 and table S1. The composite map has been deposited at EMDB-73813 (PDB: 9Z4W), the vault cap has been deposited at EMDB-73819 (PDB: 9Z5N), and the vault shell map has been deposited at EMD-73817 (table S1). Subsequent steps were performed in CryoSPARC (37) unless otherwise indicated.

Movie frames were aligned using Patch Motion in CryoSPARC. After Contrast Transfer Function (CTF) estimation using Patch CTF, low-quality micrographs with high relative ice thickness, high-motion pixel distances, and low-CTF fit resolution were removed from each dataset. Vault particles were picked using Template Picker in CryoSPARC using templates derived from initial two-dimensional (2D) classes. Low-resolution ab initio models were generated with the initial sets of particles that went through a round of 2D classification. Iterative rounds of 2D classification and heterogeneous refinement further removed particles that do not contribute to high-resolution reconstructions. A second round of particle picking was conducted with Topaz using particles manually curated from the initial selection to train the neural network (38). Particles were extracted with a box size of 700 px, binned to 1.13 Å/px, and refinement was performed with nonuniform (NU) regularization in CryoSPARC (39) with D39 symmetry enforced. The pixel size was calibrated by fitting the vault crystal structure (PDB: 4HL8) to the map at a range of nominal pixel sizes using fitmap in UCSF Chimera and identifying the pixel size resulting in the maximum cross-correlation. The particles from the initial NU refinement were subjected to reference-based motion correction (RBMC) in CryoSPARC, and resulting motion-corrected particles were Fourier cropped to 700 px. After RBMC, particles were grouped into 25 to 50 image shift groups to enable grouped refinement of beam tilt and trefoil aberrations, and NU-refine was performed again, resulting in the consensus reconstruction for the vault shell.

In the global homogeneous and NU refinement maps, the cap region of the vault appeared poorly resolved with smeared or uninterpretable density. To improve the local resolution of this region and resolve the local symmetry, local processing was performed as follows. First, local soft masks were generated using UCSF ChimeraX (36) and CryoSPARC. Then, particles were symmetry expanded using D1 symmetry. Subparticles were then extracted in a 384-px box centered on each cap region, and the per-subparticle defocus was adjusted on the basis of the calculated z-height difference with the center of the vault using a CryoSPARC Tools script. These subparticles were then subjected to local refinement with C39 symmetry using the cap-region mask. Focused classification on the cap region without alignment in C1 revealed three C13-symmetric classes distinguished by rotation around the z axis. The three classes were aligned using Align 3D maps in CryoSPARC, updating particle alignments accordingly, and subjected to local refinement using the cap-region mask, with C13 symmetry enforced. Additional 3D classification was performed, and one final round of local refinement with C13 symmetry was applied to the best class.

Atomic model building and refinement

Published vault structures [PDB: 4HL8 (40) and PDB: 7PKR (10)] were used as initial models for building. The atomic structures of vault were fitted in cryo-EM maps, and the cap region was then manually extended and completed in Coot (41). The structures and electron microscopy maps were visualized using UCSF Chimera (42) or ChimeraX. The model for the trimeric asymmetric unit was expanded into a 13x3-mer using SERVALCAT (43), followed by the refinement of the model using PHENIX Real Space Refinement (fig. S4). To aid model building of the overall vault, composite maps were generated by aligning vault cap and vault shell maps to the global reconstruction and then combining them by taking the maximum value at each voxel using UCSF Chimera. Last, models were fit to the composite map, merged, and then flexibly fit using PHENIX Real Space Refinement. Side chains of F842 and N843 were removed because of poor density. Dock prep (44) was performed for electrostatic and CHAP analysis. Figures were prepared using UCSF ChimeraX. Refinement and validation statistics are provided in table S1.

Acknowledgments

We thank Z. Zhang, R. Grassucci, and Y. Kao of the Columbia University Cryo-Electron Microscopy Center for assistance in cryo-EM grid screening and data collection. We thank Y. Guo (VCU; NCMNtech LLC) for the protocol for GO grid preparation. We also thank Z. Hu for manuscript feedback and support.

Funding:

The authors acknowledge that they received no funding in support of this research.

Author contributions:

F.V.: Conceptualization, investigation, methodology, resources, and supervision. H.L.: Writing—original draft, conceptualization, investigation, writing—review and editing, methodology, resources, data curation, validation, formal analysis, software, and visualization. O.B.C.: Writing—original draft, conceptualization, investigation, writing—review and editing, methodology, resources, data curation, validation, supervision, formal analysis, project administration, and visualization.

Competing interests:

The authors declare that they have no competing interests.

Data, code, and materials availability:

Cryo-EM maps and structures have been deposited in the PDB and EMDB databases, respectively, with the following accession codes: 9Z5N and EMD-73819 (vault cap), 9Z4W and EMD-73814 (composite map and model), and EMD-73817 (D39 consensus reconstruction of vault). All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. This study did not generate new materials.

Supplementary Materials

The PDF file includes:

Figs. S1 to S5

Table S1

Legend for movie S1

sciadv.adz6794_sm.pdf (8.9MB, pdf)

Other Supplementary Material for this manuscript includes the following:

Movie S1

sciadv.adz6794_movie_s1.zip (20.8MB, zip)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S5

Table S1

Legend for movie S1

sciadv.adz6794_sm.pdf (8.9MB, pdf)

Movie S1

sciadv.adz6794_movie_s1.zip (20.8MB, zip)

Data Availability Statement

Cryo-EM maps and structures have been deposited in the PDB and EMDB databases, respectively, with the following accession codes: 9Z5N and EMD-73819 (vault cap), 9Z4W and EMD-73814 (composite map and model), and EMD-73817 (D39 consensus reconstruction of vault). All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. This study did not generate new materials.

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