Recombinant human CP expression

Recombinant systems have enabled the study of mature proteasome complexes20,58,59,60. To express human CP in insect cells, we used a combination of biGBac61 and MultiBac62 systems to generate baculovirus transfer vectors: one with all seven α subunits, another with all seven β subunits and a third with the five CP assembly chaperones. For affinity purification, we appended a C-terminal twin-Strep tag to either β2 or β7, the first and last CP subunits, respectively, to incorporate. We performed affinity purification on CP, followed by size-exclusion chromatography (SEC) (Fig. 1a,b). Both preparations showed the expected electrophoretic properties for mature CP. Furthermore, cryo-EM analyses of the peak fractions from both preparations yielded high-resolution structures that superimposed with native human 20S CP (root-mean-square deviation (r.m.s.d.) 0.703 Å) (Fig. 1c, right, and Extended Data Figs. 1a–e and 2).

Fig. 1: Recombinant expression of human 20S proteasome in insect cells.

a, Purification of recombinant β2-tagged (PSMB7) 20S CP. Eluates were subjected to SEC, and the fractions were analyzed by SDS–PAGE, followed by Coomassie staining. The asterisk indicates bands in the later fractions (corresponding to complexes smaller than a mature 20S CP) of lower molecular weight than proteasome subunits that would correspond to the assembly chaperones PAC1–PAC4 and POMP. Red, blue and purple dots indicate fractions used for cryo-EM, with the later-migrating fraction (blue dot) yielding high-resolution structures 1–5 of 20S CP assembly intermediates and the early fraction (red dot) yielding structures of β2-tagged preholo, premature and mature 20S CP. MW, molecular weight; mAU, milli-absorbance units. b, Purification of recombinant β7-tagged (PSMB4) 20S CP. The single-peak fraction (purple dot) yielded a high-resolution structure of β7-tagged 20S CP. c, Cryo-EM maps of eight distinct CP subcomplexes. Their subunits, the chaperone configurations and the resolved region in each map are indicated. Check marks and cross marks indicate subunits that are visible or absent in the corresponding maps, respectively. Similar results for a and b were obtained in three independent experiments.

Source data

The SEC profile for the β2-tag purification revealed a shoulder peak consistent with lower-molecular-weight subcomplexes (Fig. 1a). SDS–PAGE analysis revealed additional proteins with a size of 12–15 kDa. Some assembly chaperones migrate in this size range, suggesting that these complexes might represent immature CP species. Cryo-EM analysis of these fractions yielded structures of five distinct immature CP subcomplexes with overall resolutions ranging from 2.67 to 2.95 Å (Fig. 1c, left, Extended Data Figs. 3 and 4 and Table 1). We order these structures on the basis of the number of visible β subunits, with their compositions indicated in Fig. 1c. We present the structures with α subunits in blue and β subunits in purple, increasing in shade by subunit number (α-ring) or order of incorporation (β-ring). These structures are consistent with the ordered, sequential addition of β subunits proposed from genetic knockdown studies in human cells42.

Table 1 Cryo-EM data collection, refinement and validation statistics

Structure 1 represents an early CP subcomplex and consists of the α-ring, all five chaperones and β2 (Fig. 1c and Supplementary Video 1). Structures 2–5 consist of the α-ring, chaperones POMP and PAC1/PAC2, and increasing numbers of β subunits (Fig. 1c and Supplementary Videos 2–5). In addition to mature CP, detailed analysis of the cryo-EM data from the higher-molecular-weight fraction revealed two supra-20S structures, referred to as ‘preholo 20S CP’ and ‘premature 20S CP’ (Fig. 1c, right, and Extended Data Fig. 1d–f). Structures 3 and 4 are analogous to recent structures of yeast 13S and pre-15S intermediates, respectively56. Meanwhile, cryo-EM maps of various resolutions have also shown constituents of yeast preholo 20S and premature 20S CP complexes63,64. Thus, the structural progression of human complexes lies within a conserved proteasome assembly pathway. Comparing the seven different structures to each other and to matched mature CP allows visualizing the conformational progression across the assembly pathway (Supplementary Video 6) and the specific molecular interactions occurring at each step, as described below.

Chaperones coordinate α-ring formation and β-ring initiation

Structure 1 shows PAC1/PAC2 and PAC3/PAC4 within the same complex and illustrates POMP-dependent initiation of β-ring assembly through its binding of both the α-ring and β2 (Supplementary Video 1). Together, PAC1/PAC2 and PAC3/PAC4 bind all seven α subunits (Fig. 2a,b, zoomed-out views). PAC1/PAC2 is perched atop the α-ring (Fig. 2a), on the surface recognized by proteasome activators. This placement appears to serve two main purposes. First, it likely helps coordinate α-ring configuration because PAC1/PAC2 contact all α subunits except α3 (Fig. 2c). Second, as observed for the yeast counterpart Pba1/Pba2 in later proteasome assembly intermediates27, PAC1/PAC2 directly bind the N termini from multiple α subunits (Fig. 2a, close-up) and, through numerous interactions, orchestrate an open-gate conformation (Fig. 2d–g). Perhaps most dramatically, PAC1/PAC2 bind α5’s N terminus more than 20 Å away from the α-ring (Fig. 2a and Supplementary Video 1). This interaction likely contributes to PAC1/PAC2’s reported ability to bind α5 in isolation35.

Fig. 2: Structural progression of early assembly intermediates.

Cryo-EM maps of eight distinct CP subcomplexes; opaque images represent complexes described in the figure. a, Cross-section side view of cryo-EM map of structure 1 showing all five assembly chaperones. PAC1/PAC2 are perched atop the α-ring and interact with all α subunits except for α3. term, terminus. b, Bottom-up view of cryo-EM map of structure 1 showing PAC3/PAC4 in the groove between α4–α5 and α5–α6 and β2 in the groove between α1 and α2. c, Top-down view of map 1 showing the interaction of PAC1/PAC2 with the α-ring; the central pore is open. d, Close-up view of the HbYX motif of Rpt5 inserted in between α5 and α6 in native substrate-bound 26S proteasome (PDB 6MSJ). e, Close-up view of the HbYX motif of PAC1 inserted between α5 and α6 in structure 1. f, Close-up view of the HbYX motif of PAC2 inserted between α6 and α7 in structure 1. g, Top-down view of structure 1 (PAC1/PAC2 not shown) showing that the α-ring ‘gate’ is open. h, Side view of structure 1 showing that PAC3/PAC4 interact with α3–α7 of the α-ring. i, Side view of structure 1 showing that POMP interacts with α1–α3 and α7 of the α-ring. j, Cross-section view of cryo-EM map of structure 2 showing that the incorporation of β3 is mutually stabilized by the β2 propeptide (propep) and β2 CTE. Close-up view: the additional POMP N-terminal region interacting with the β2 propeptide and β3. The β2 propeptide is colored green. Most of POMP is colored orange, but the designated regions are yellow. k, Close-up view of the PAC1 N terminus of structure 2 showing its interaction with α1 and POMP. l, Left: bottom-up view of cryo-EM map of structure 2 showing that the release of PAC3/PAC4 liberates grooves between α4–α5 and α5–α6 and that β3 occupies the α2–α3 groove. Right: top-down view showing that the pore is closed. m, Cross-section view of cryo-EM map of structure 3 showing the incorporation of β4 and its interaction with β3 and the α-ring.

The visualized N termini from α subunits have features in common with those in substrate-engaged 26S proteasome65 (Supplementary Video 7), which shows an open gate. This arrangement sharply contrasts with the mature CP, wherein the N termini from α subunits extend into and block access to the central pore2. Similar to known proteasome activators such as the RP (Fig. 2d)12,15,19,20,21,66, PAC1 contains a canonical HbYX motif (Ile-Tyr-Thr)48,56, which inserts into the α5–α6 pocket (Fig. 2e). PAC2 contains a variant sequence (Leu-Phe), with Phe positioned in the α6–α7 pocket similarly to conventional interactions with the Tyr residue from the HbYX motif (Fig. 2f).

PAC3/PAC4 sit on the opposite face of the α-ring (that is, the future site of β4–β7 subunits later during assembly) (Fig. 2a,b and Supplementary Videos 1 and 6). Consistent with the structure of the orthologous isolated yeast Pba3/Pba4–α5 complex (Extended Data Fig. 5a)47,67,68, PAC3/PAC4 occupy the α3–α7 side of the ring, with α5 binding at the PAC3/PAC4 interface (Fig. 2b). By occupying these sites, PAC3/PAC4 not only stabilize the α-ring to provide a platform for β-ring assembly, but also preclude incorporation of the late β subunits, helping to order the incorporation of β subunits.

Unexpectedly, PAC3/PAC4 and the β-ring chaperone POMP directly interact (Fig. 2a,b). The same site in POMP bound by PAC3 is recognized later during assembly by the β5 propeptide (Extended Data Fig. 5b and Supplementary Videos 6 and 8). POMP consists of conserved helices and loops that become progressively more resolved upon the successive addition of β subunits, with POMP ultimately winding back and forth between the α-rings and β-rings (Extended Data Fig. 5c and Supplementary Video 8). In structure 1, one end of POMP is enclosed by PAC3, α7 and α1 (Fig. 2h), with the other end enclosed by PAC4, α3 and α4 (Fig. 2i). These contacts rationalize previous observations of POMP’s direct interaction with the α-ring in isolation and with α3, α4 and α7 according to a yeast two-hybrid assay42,69.

Lastly, structure 1 reveals β-ring initiation with β2; a PAC3/PAC4-bound complete α-ring engages POMP, which co-recruits β2 (Supplementary Video 1). The well-resolved portion of β2 is that inserted between α1 and α2 and in contact with POMP (Fig. 2h). The distal regions of β2 are poorly resolved, presumably due to flexibility without constraint from adjacent β subunits.

The β2 propeptide and POMP secure β3 and β4

Structure 2 is distinguished by the loss of PAC3/PAC4 and addition of β3 (Fig. 2j,k and Supplementary Videos 2 and 6), and it explains previous observations from genetically engineered mammalian cells that β3 is the second β subunit to enter and this requires prior β2 addition42. The β2 subunit grasps β3 with both a C-terminal extension (CTE) and the β2 propeptide (Fig. 2j), rationalizing why β3 incorporation in mammalian cells requires these β2 elements42. Extending from the β2 active site Thr, the propeptide meanders, forms a helix crossing β3’s interior surface and extends to secure newly visible regions of POMP that co-fasten β3 in the groove between α2 and α3 (Fig. 2j, bottom left). Meanwhile, part of β2’s CTE forms a strand that extends a β-sheet from the adjacent β3, similar to the arrangement seen in mature CP2,70.

β3 is stabilized by newly visible residues of POMP (residues 45–61 and 114–141) in structure 2 compared to structure 1 (Fig. 2j). Residues 114–141 directly clamp β3 against the α-ring through an interaction that is also buttressed by part of the β2 propeptide (Fig. 2j, bottom). POMP residues 45–61 buttress part of the β2 propeptide that secures the interior surface of β3 (Fig. 2j, bottom). Notably, the regions of the β2 propeptide and POMP that clamp β3 in place bind the same α3 and α4 surfaces as PAC3/PAC4 did in structure 1 (Fig. 2a,j, zoomed-out views, and Supplementary Video 6). This suggests that PAC3/PAC4 eviction may coincide with the stabilization of those regions of POMP and the β2 propeptide upon incorporation of β3. This could explain why β3 knockdown causes accumulation of the five-chaperone-containing intermediate42 even though β3 does not directly overlap with PAC3/PAC4.

Upon departure of PAC3/PAC4, α-ring stability may be alternatively supported by POMP bridging β2–β3 and the α-ring, as well as changes at the α-ring pore. The N termini of α1 and PAC1 are newly observed in structure 2, passing through the gate toward POMP (Fig. 2k and Supplementary Video 2). PAC1’s N terminus runs alongside α1’s N terminus through the center of the α-ring until PAC1 contacts a newly ordered region of POMP and makes a U-turn to terminate in the center of the α-ring pore (Fig. 2k,l and Supplementary Video 2). Concomitantly, additional N-terminal residues of α2–α4 become visible, stabilized in an open conformation by a collar of Tyr residues between α-ring subunits rather than by direct, extensive interactions with PAC1/PAC2, as occurs for α5 (Extended Data Fig. 5d). This open-gate but occluded pore arrangement is maintained throughout the other assemblies we describe, including the preholo 20S CP.

Key differences between structures 1 and 2 set the stage for the β4 incorporation seen in structure 3 (Fig. 3a and Supplementary Video 3). One side of β4 binds a composite surface from β3 and the β2 propeptide, while its other side binds the α4 region that was bound to PAC4 in structure 1 (Fig. 2a,m and Supplementary Video 6). Importantly, structure 3 corresponds to the well-recognized 13S precursor39,55 and is largely superimposable on yeast 13S56, although there are notable differences accommodating the divergent PAC1 and yeast Pba1 N termini. First, while the N termini of both PAC1 and Pba1 thread through the open gate into the CP interior, in yeast, the N terminus of α2 (ref. 66), not α1, runs alongside Pba1’s N terminus (Extended Data Fig. 5e,f). Second, PAC1’s longer N terminus loops back upward toward the α-ring gate, whereas Pba1’s N terminus ends in contact with Ump1 and the β5 propeptide56 (Extended Data Fig. 5g).

Fig. 3: Distinct structural features upon β5 and β6 incorporation.

Cryo-EM maps of eight distinct CP subcomplexes; opaque images represent complexes described in the figure. a, Cross-section view of cryo-EM map of structure 3 showing PAC1/PAC2, POMP and β2–β4. Left: close-up view of POMP showing its helix–turn–helix configuration and its interactions with the α-ring and β subunits. Right: close-up view revealing the interaction between POMP and the β2 propeptide. b, Cross-section view of cryo-EM map of structure 4 showing PAC1/PAC2, POMP and β2–β5 upon β5 and β6 incorporation (β6 is cropped in the cross-section). Left: close-up view of POMP showing that the additional POMP N-terminal region (blue) is resolved and stabilizes β5 and β6 incorporation. Right: close-up view of the β2 propeptide (green) revealing that the additional resolved POMP N-terminal region interacts with and reorients the N terminus of the β2 propeptide. c, Close-up view of β5, β6 and POMP showing that the additional resolved N-terminal region of POMP interacts with the β5 loop. The β5 propeptide is colored yellow. d, Close-up view of the β5 propeptide showing its interaction with α6, α7, α1 and POMP.

A POMP–propeptide network internally scaffolds the β-ring

Structure 4 corresponds to the yeast pre-15S intermediate56 and contains the α-ring, β2–β6, PAC1/PAC2 and POMP, while structure 5 also contains β1 and β7 and shows better resolution of β6 (Supplementary Videos 4 and 5). These structures illustrate how portions of assembly factors become progressively structured as they secure the remaining β subunits (Supplementary Video 8 and Extended Data Figs. 6 and 7). Insertion of β5 and β6 is accompanied by increased visualization of POMP’s N terminus (residues 29–44) (Fig. 3a,b, left) accompanied by redirection of the β2 propeptide’s N terminus to fill a newly formed pocket at the confluence of POMP, β4–β5 and α3–α4 (Fig. 3a,b, right).

In structure 4, only the extreme N terminus (residues 2–8) of the β5 propeptide is visible, at the junction of α6, α7 and POMP. This resembles a harpoon, anchored 60 Å away from the rest of β5 (Fig. 3c,d and Supplementary Video 4). Structure 5 shows the assembly factors within a completed β-ring just before half-CP fusion (Supplementary Video 5). Additional elements in the β5 propeptide and POMP form a belt supporting the β5–β1 subunits (Fig. 4a,c). Broader regions of the β5 propeptide are newly visible in structure 5 compared to structure 4, suggesting that its folding is coordinated with the progressive addition of β subunits (Fig. 4a,b and Supplementary Videos 6 and 8). After emerging from mature β5, the β5 propeptide winds back and forth between the β-ring and α-ring to secure β6, β7 and β1 and then crosses POMP before terminating with the interactions already observed in structure 4 (Fig. 4b,c). The β5 propeptide’s interactions at β6’s interface with the α subunits clarify observations that the β5 propeptide is essential for the incorporation of β6, while β5 does not require its own propeptide for incorporation42. Meanwhile, newly visible N-terminal regions of POMP in structure 5 support the β6–β5 and β1–β2 pairs of subunits (Fig. 4b, right). A portion of the β1 propeptide (residues 16–34), visualized in structure 5, resembles a staple extending from β1 to β7 (Fig. 4d).

Fig. 4: Incorporation of β7 and β1 completes the β-ring and reveals propeptides from all proteolytic subunits.

Cryo-EM maps of eight distinct CP subcomplexes; opaque images represent complexes described in the figure. a, Cross-section view of cryo-EM map of structure 4 showing PAC1/PAC2, POMP, β3 and β6 upon β5 and β6 incorporation (β2 is hidden behind, and β4–β5 are cropped in the cross-section). Left: close-up view of β-ring. Right: interaction between β5 propeptide and POMP. The β2 propeptide is colored green, and the β5 propeptide is colored yellow. b, Cross-section view of cryo-EM map of structure 5 showing PAC1/PAC2, POMP, β3 and β6 upon β7 and β1 incorporation (β7 and β1–β2 are hidden behind, and β4–β5 are cropped in the cross-section). Left: close-up view showing that the additional β5 propeptide is resolved upon completion of the β-ring compared to β5 in structure 4. Right: close-up view of the β5 propeptide’s interaction with POMP, β1 and β2 showing that POMP’s N terminus (blue) is resolved upon completion of the β-ring and interacts with the β5 propeptide. The β2 propeptide is colored green, and the β5 propeptide is colored yellow. c, Close-up view of the β5 propeptide’s interaction with α5–α7, β5–β6 and β1. d, Close-up view of newly incorporated β1 and β7 revealing the β1 propeptide and its interaction with the β7 N terminus and loop. The β1 propeptide is colored cyan.

Half-CP fusion and proteolytic activation

Comparing the half-CP in structure 5 and the preholo, premature and mature 20S CP structures reveals conformational changes accompanying half-CP fusion and eventual loss of propeptides, POMP and PAC1/PAC2 during 20S proteasome maturation (Fig. 5a and Supplementary Video 9). The most striking differences between structure 5 and preholo 20S CP are in the conformations of the β subunits. Fusion of two half-CPs coaxes the proteases toward active conformations (Fig. 5b–e). In particular, two key elements from each subunit along the β-ring–β-ring interface are a β-hairpin (for example, residues 61–75 of β2) and adjacent loop (for example, residues 205–215 of β2) (Fig. 5c). We refer to these as ‘fusion hairpins’ and ‘fusion loops’, respectively, because they interlock in a zipper-like structure with those from the opposing subunit across the β-ring–β-ring interface (Extended Data Fig. 8a–h). However, in the half-CP (structure 5), these elements are exposed, and either the majority are not visible and presumably dynamic (β2, β3, β5 and β1) or the β-hairpin is splayed outward or inward relative to the subunit center (β4 and β7) (Extended Data Fig. 8b–h). The fusion hairpins and fusion loops become visible and/or are reoriented upon fusion, as seen in the preholo intermediate, where their conformations resemble the mature CP (Extended Data Fig. 8i–k).

Fig. 5: Half-CP fusion and 20S CP maturation.

Cryo-EM maps of eight distinct CP subcomplexes; opaque images represent complexes described in the figure. a, Cross-section view of cryo-EM map of structure 5, preholo 20S CP, premature 20S CP and mature 20S CP showing the earlier presence and subsequent absence of assembly chaperones and β propeptides during the 20S CP fusion and maturation process. b, Cryo-EM map of structure 5 with β1, β2 and β5 colored in yellow. Cryo-EM map of preholo 20S CP with β1, β2 and β5 of one half-CP colored in purple and the other half-CP colored in salmon. c, Close-up view of β2’s structural changes (β2 from structure 5 colored in yellow and β2 from preholo 20S CP colored in purple) and interactions with β6 (salmon) from the opposite half-CP upon CP fusion. The active site position is shown and fusion hairpin and fusion loop elements are labeled ‘hairpin’ and ‘loop’, respectively. d, Close-up view of β5’s structural changes (β5 from structure 5 colored in yellow and β5 from preholo 20S CP colored in purple) and interactions with β3 (salmon) from the opposite half-CP upon CP fusion. The active site position is shown and fusion hairpin and fusion loop elements are labeled ‘hairpin’ and ‘loop’, respectively. e, Close-up view of β1’s structural changes (β1 from structure 5 colored in yellow and β1 from preholo 20S CP colored in purple) and interactions with β7 (salmon) from the opposite half-CP upon CP fusion. The active site position is shown and fusion hairpin and fusion loop elements are labeled ‘hairpin’ and ‘loop’, respectively.

Overall, the structural similarity of β1, β2 and β5 between the preholo 20S intermediate and mature 20S CP is consistent, with fusion having a major role in driving autocatalytic activation. When ordered after fusion, the fusion hairpins and fusion loops abut the catalytic Thr residues of β1, β2 and β5, and they shape the active sites of the protease subunits (Fig. 5c–e). Furthermore, the fusion loop contains key residues required for autocatalytic protease activation53.

For the β2 subunit, there is additional restructuring of the protease domain. In structures 3–5, β2’s residues 225–237 face the α-ring and cling to the adjacent α2 subunit (Fig. 5c). This interaction was not observed in proteasome assembly intermediates from yeast because of this α2 region’s different sequence and structure (Extended Data Fig. 8l). For human β2, the post-fusion structures show residues 225–237 making a U-turn and proceeding toward the other half-CP (Fig. 5c, middle). This structural remodeling, commensurate with the fusion of two half-CPs, completes β2’s protease fold. This conformational change also directs subsequent β2 residues (237–247) toward the opposite half-CP. These β2 residues were not visible and presumably dynamic in the structures across β-ring assembly, but, after fusion, they formed a belt engaging β6 from the opposite half-CP (Fig. 5c, bottom).

Interestingly, in the preholo 20S CP structure, density corresponding to the propeptides continues from the β2 and β5 active sites (Extended Data Fig. 8m,n). Meanwhile, prior crystallographic and functional analyses of yeast proteasomes harboring variations in the propeptides and residues adjacent to the active site suggested that the rate of propeptide cleavage is influenced by even subtle perturbation of (1) backbone conformation at the junction between the active site and propeptide; (2) adjacent side chains including a critical Lys; and (3) the constellation of surrounding water molecules40,53. Although the resolution of our structures precludes precise placement of atoms and visualization of water molecules, the preholo 20S CP intermediate structure shows features poised to impact these facets of propeptide maturation. First, the β2 and β5 propeptides make numerous contacts that impact their orientation. For the β2 propeptide, these mirror those in structure 5 and include interactions with β3, β4, α3 and POMP (Extended Data Fig. 8m). Meanwhile, residues 52–59 of the β5 propeptide retain contacts with β6 (Fig. 5d). Second, the propeptides align their active site and adjacent residues critical for their cleavage53 (Fig. 5c–e).

Although the human preholo 20S CP exhibits two-fold symmetry and maintains much of the assembly factor scaffold, only a single PAC1/PAC2 is visible in the premature complex (Fig.