Structure of the 26S proteasome fromSchizosaccharomyces pombe atsubnanometer resolutionStefan Bohna, Florian Becka, Eri Sakataa, Thomas Walzthoenib, Martin Beckb,1, Ruedi Aebersoldb,c, Friedrich Förstera,Wolfgang Baumeistera,2, and Stephan Nickella

aMolecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; bInstitute of Molecular Systems Biology, EidgenössischeTechnische Hochschule, 8093 Zürich, Switzerland; and cFaculty of Science, University of Zürich, 8093 Zürich, Switzerland

Contributed by Wolfgang Baumeister, October 19, 2010 (sent for review September 16, 2010)

The structure of the 26S proteasome from Schizosaccharomycespombe has been determined to a resolution of 9.1 Å by cryoelec-tron microscopy and single particle analysis. In addition, chemicalcross-linking in conjunction with mass spectrometry has been usedto identify numerous residue pairs in close proximity to each other,providing an array of spatial restraints. Taken together these dataclarify the topology of the AAA-ATPase module in the 19S regula-tory particle and its spatial relationship to the α-ring of the 20Score particle. Image classification and variance analysis reveal abelt of high “activity” surrounding the AAA-ATPase module whichis tentatively assigned to the reversible association of proteasomeinteracting proteins and the conformational heterogeneity amongthe particles. An integrated model is presented which sheds lighton the early steps of protein degradation by the 26S complex.

deubiquitylating enzymes ∣ macromolecular complex ∣ ubiquitin-proteasome pathway ∣ ubiquitin receptor ∣ single particle classification

In eukaryotic cells, most proteins in the cytosol and the nucleusare regulated via the ubiquitin-proteasome pathway and

malfunctions of this pathway have been implicated in a wide vari-ety of diseases (1). The 26S proteasome is the most downstreamelement of this pathway, executing protein degradation (2–4).Unlike constitutively active proteases, the proteasome has thecapacity to degrade almost any protein, yet it acts with exquisitespecificity. The key stratagem is self-compartmentalization: Theactive sites of the proteolytic 20S core particles (CPs) are seques-tered from the cellular environment in the interior of this barrel-shaped subcomplex (5). Proteins destined for degradation aremarked by a polyubiquitin chain, a degradation signal that isrecognized by the 19S regulatory particles (RPs) that bind toeither one or both ends of the CP to form the 26S holocomplex.The RPs (i) recognize the polyubiquitylated substrates, (ii) trimand recycle the polyubiquitin chains, (iii) unfold substrates to bedegraded, and (iv) open the gate to the CP and assist in substratetranslocation into the interior of the CP. These tasks are per-formed by a complex machinery involving at least 19 different sub-units, 6 AAA-ATPases (Rpt1–6), and 13 non-ATPases (Rpn1–3,Rpn5–13, Rpn15/Sem1p).

Although the structure of the CP has been elucidated in greatdetail by X-ray crystallography (6, 7), the structure of the RP isonly dimly understood at present. Best characterized are theAAA-ATPases which form a heterohexameric subcomplex situ-ated at the base of the RP in close proximity to the α-rings ofthe CP (8, 9). The C-terminal residues of Rpt2 and Rpt5 wereshown to be involved in opening the gate in the α-rings, allowingsubstrates to enter the CP. A similar mechanism has been postu-lated for proteasome-activating nucleotidase (PAN), the archaealhomohexameric homolog of the eukaryotic AAA-ATPase module(10). Crystal structures of the two major fragments of PANsuggest that the N- and C-terminal domains form two stackedconcentric rings (N ring and AAA ring) (11, 12); the N ring is

implicated in substrate unfolding and the AAA ring in gate open-ing and substrate translocation.

Among the non-ATPases, the functions of Rpn10, Rpn11, andRpn13 are well known. Rpn11 is a deubiquitylating enzyme andresponsible for ubiquitin (Ub) removal from substrates and Ubrecycling (13, 14), whereas Rpn10 and Rpn13 are Ub receptors(15). Their localization within the RP would be particularly infor-mative in terms of understanding the sequence of events betweenthe initial binding of substrates and their translocation intothe CP.

Given the complexity of the 26S proteasome and its fragilenature, its dynamics and the association-dissociation of protea-some interacting proteins (PIPs) and, therefore, their presence invariable amounts, it has been impossible so far to obtain crystalssuitable for a high-resolution structural analysis by X-ray crystal-lography. For cryoelectron microscopy (cryo-EM) and forprotein–protein interaction studies, the requirements for samplehomogeneity are less stringent. A ∼25-Å resolution structure ofthe Drosophila melanogaster 26S proteasome has been reportedrecently (9), revealing the basic organization of the RP anddefining, in particular, the localization and the boundaries of theAAA-ATPase module.

Here we present a structure of the 26S complex isolated fromSchizosaccharomyces pombe at much higher resolution [9.1 Å atFourier shell correlation (FSC) of 0.5 and 6.7 Å at FSC of 0.3].Moreover, we have used chemical cross-linking in conjunctionwith MS to identify numerous residue pairs in close proximityto each other providing us with an array of spatial restraints(16–18). The integration of these data into the medium resolutionEM maps allowed us to generate a model providing insights intothe structural organization of the 26S holocomplex.

Results and DiscussionPurification and MS Analysis. We analyzed the purified S. pombe26S proteasomes by quantitative MS (Fig. S1). The canonical 26Sproteasome subunits α1–7, β1–7, Rpt1–6, Rpn1–3, and Rpn5–12are all present in equimolar amounts. Rpn13 is present in twodifferent orthologs, Rpn13a (Uniprot ID Q9Y7Y6) and Rpn13b(Uniprot ID Q9USM1), each in a ∼0.2∶1 ratio. Expression oforgan-specific Rpn13 orthologs was previously reported forD. melanogaster testis (19), but simultaneous expression of differ-ent orthologs has not been described in other species. The abun-dance of the 9 kDa peptide Rpn15 could only be determined with

Author contributions: S.B., F.B., E.S., R.A., F.F., W.B., and S.N. designed research; S.B., F.B.,E.S., T.W., M.B., and S.N. performed research; S.B., F.B., E.S., T.W., and M.B. analyzed data;and S.B., F.B., E.S., T.W., M.B., R.A., F.F., W.B., and S.N. wrote the paper.

The authors declare no conflict of interest.1Present address: European Molecular Biology Laboratory Heidelberg, 69117 Heidelberg,Germany.

2To whom correspondence should be addressed. E-mail: baumeister@biochem.mpg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1015530107/-/DCSupplemental.

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low accuracy by our MS approach because only one unique pep-tide was identified. The ubiquitin C-terminal hydrolases Uch2and Ubp6, as well as Ub were detected in significant amounts.

When comparing the relative stoichiometry of the S. pombe26S proteasome to that determined for Drosophila (9) we foundthe following differences: Rpn10 is substantially more abundantin S. pombe (∼0.8∶1) than in theDrosophila (∼0.25∶1). Moreover,Rpn13a/Rpn13b and Uch2 are present in substoichiometricamounts in S. pombe, whereas Rpn13 and Uch37 were foundin stoichiometric amounts in Drosophila proteasomes. Interest-ingly, in 26S proteasomes purified from Homo sapiens, none ofthese subunits (Rpn10, Rpn13, Uch2/Uch37) are present in sig-nificant amounts (20). Thus, quantitative analysis indicates thatα1–7, β1–7, Rpt1–6, Rpn1–3, Rpn5–9, and Rpn11–12 constitutethe canonical core of the 26S proteasome, whereas the subunitsRpn10 and Rpn13, albeit always present, are found in variableamounts.

The 26S Proteasome at Subnanometer Resolution. From vitrifiedsamples of our 26S preparation, micrographs were acquired ina semiautomated fashion. Micrographs typically showed com-plete double-capped 26S proteasome particles, but some singlecapped particles, as well as isolated CPs were also observed(Fig. S2). From 20,000 micrographs, we selected 270,000 holo-complexes for single particle analysis.

Due to the fragility and dynamics of the 26S proteasome, weanticipated substantial structural variability among individualparticles. To “purify” the particle ensemble in silico we used amaximum likelihood-based classification method (ML3D) (21)and classified the particles into two different groups (Fig. S3).One class showed all the features of 26S holocomplexes, whereasthe other clearly corresponded to partially (dis)assembled 26Sproteasomes (∼85;000 particles). In a second ML3D classifica-tion step, we were able to further separate the holocomplexes into26S proteasomes which differed by a distinct mass in the cap re-gion (“classD1” with one and “classD2” with two “extra masses”containing ∼85;000 and ∼100;000 particles, respectively).

The variable mass was at approximately the same positionwhere a variable mass was observed before with the D. melano-gaster proteasome (9). However, in the present study occupancywith the extra mass was much higher than in D. melanogaster.Whereas in S. pombe the extra mass was present at either oneor both RPs, the extra mass was observed only at one RP ornot at all in the D. melanogaster proteasome (Fig. S4). In D. mel-anogaster, the relative abundance of the extra mass compared tothe remaining RP density (∼25%) correlated to the abundance ofRpn10 as determined by MS. Therefore, Rpn10 was tentativelylocalized to the extra mass. Interestingly, in the present study, therelative abundance of the extra mass (∼75%) also correlates tothe Rpn10 stoichiometry (0.8∶1). Thus, our EM and MS datasupport the putative localization of Rpn10.

The 3D densities corresponding to intact holocomplexes dis-played C2 symmetry (apart from the extra mass). Therefore,to obtain higher resolution, we imposed C2 symmetry for the re-construction of the 26S proteasome density from all holocom-plexes (resolution of 9.1 and 6.7 Å at FSC of 0.5 and 0.3,respectively; Fig. S5). In the final symmetrical reconstruction,several helical motifs became discernible (Fig. 1A, Fig. S6, andMovie S1).

We fitted a comparative model of the CP into the EM map(Fig. 1B). The prominent N-terminal α-helix, a hallmark of theα4 subunit, allows unambiguous positioning of the CP. The fitof the atomic model and the EM map is excellent, as indicatedby the high cross-correlation coefficient (CCC ¼ 0.78, both mod-els filtered to 9 Å) and the visual correspondence of helices inmodel and map. The density at the gate of the CP is in goodagreement with the closed-state of the crystal structure.

Adjacent to the CP, we positioned a comparative model of theAAA-ATPases Rpt1–6 into the EM map as described previously(22) (Fig. 1). The C-terminal AAA-ATPase domains form theAAA ring that binds to the CP, whereas the N-terminal segmentsform a smaller ring positioned atop of the AAA ring (11, 12). Inthe EMmap, density corresponding to the N-terminal coiled coilsis clearly discernible and correlates well to the atomic model. Theatomic model of the AAA ring is in good agreement with the EMdensity, albeit some helical densities do not colocalize preciselywith the helices in the atomic model indicative of dynamic con-formation changes (see below). The center of the hexamericAAA-ATPase is shifted by ∼20 Å from the pseudo-sevenfoldrotational symmetry axis of the CP, similar as observed for theD. melanogaster 26S proteasome (30 Å). The hexamer is less in-clined with respect to the CP axis; whereas the pseudo-threefoldsymmetry axis of the AAA-ATPase hexamer was tilted by ∼10°with respect to the CP axis in the D. melanogaster density, it istilted by ∼4° in the map from S. pombe.

In the remaining density of the RP, features reminiscent ofshort bihelical repeats in supercoiled quaternary structures arerecognizable (Fig. 1A, *). Indeed, such repetitive motifs, probablysimilar to tetratricopeptide repeats (TPRs), have been predictedbefore for Rpn1 and Rpn2, as well as the proteasome, COP9 sig-nalosome, eIF3 module containing subunits Rpn3, Rpn5, Rpn6,Rpn7, Rpn9, and Rpn12 (8, 23–26). The width of the helical rib-bons is ∼25 Å, which is consistent with the typical helix lengthobserved in TPRs (Fig. S6).

The stoichiometries determined by our MS analysis suggestconfigurational variation among individual particles beyondthose that can be resolved by ML3D classification. To visualizethe spatial distribution of the major variations in the 26S protea-some, we calculated a 3D variance map (27) (Fig. 1C). As ex-pected, the variance is low in the density corresponding to the CP.The lid region of the RP also shows a low variance level. Thevariance is highest in a belt surrounding the AAA-ATPase in theRP. It is known from structural studies that AAA-ATPases typi-cally undergo large conformational changes as part of their ATPcycle (28). However, the structural variation extends to areasadjacent to the hexameric AAA-ATPase. These structuralchanges can probably be mostly attributed to the substoichiome-trically bound proteins Rpn10, Rpn13a/b, and Uch2. Indeed,when comparing a reconstruction of 26S proteasomes from anUch2 deletion strain to the wild-type map, we could not observesignificant differences, which is probably due to the fact that theregion of Uch2 binding is highly variable.

Classification of the RP. To obtain further insights into the different26S proteasome conformations we grouped the particles intodifferent classes to localize structural variations specifically inthe belt around the AAA-ATPases (Fig. S7). Classes derived fromfocused classification (3D) are consistent with results obtainedfrom 2D analysis (Fig. S8). The analysis further resolves the extramass: This mass adopts a variety of different shapes, e.g., itprotrudes to the lid in one class (Fig. 2). This result would beconsistent with Rpn10 positioned in the extra mass becausethe ubiquitin interacting motif is highly flexible (29). Otherclasses show significant extra densities (of up to 30–40 kDa) ina region around the AAA-ATPases (e.g., Fig. 2, Fig. S9,Movie S2). The most likely candidates for these extra densitiesare Rpn13a, Rpn13b, Ub, and Uch2. Taken together, the variancemap and the classification results suggest that the distal part ofthe lid provides a mostly invariable frame, whereas the base andthe base–lid interface are sites of high variability. The structuralvariation (activity) is caused by both structural flexibility ofconstitutive subunits as well as the reversible association of addi-tional densities, tentatively assigned to PIPs.

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Localization of Rpn11. Using the C-terminal 3x-FLAG-epitope ofRpn11, we labeled Rpn11 with an anti-FLAG antibody (Ab)(Fig. 3). We acquired images of 26S proteasome incubated withthe anti-FLAG Ab by cryo-EM. Micrographs displayed typical26S proteasome “twin” configurations (i.e., two holocomplexesaligned with their long axes), as well as isolated 26S proteasomes(Fig. S2). The 2D class averages clearly indicate a distinct densityabove the lid region, which is not present in the unlabeled pro-teasome (Fig. 3A). Because the twins adopt a single spatial orien-tation in ice, we reconstructed single 26S proteasomes to map theAb density in 3D. Comparison of the 17.4-Å resolution map withthe map of the unlabeled 26S proteasome reveals a rather blurredadditional density indicative of a high degree of flexibility(Fig. 3B). This density can be mainly attributed to the structuralvariability of the 17-residue C-terminal linker to the 3x-FLAGepitope, the presence of three FLAG epitopes, and the intrinsicvariability of the Ab. Thus, the Ab density maps the C terminus ofRpn11 with relative low resolution: We estimate that the Rpn11C terminus is located within a radius of ∼80 Å around the centerof the Ab mass (Fig. 3C).

To obtain further data on protein–protein proximity, we sub-jected the sample to cross-linking with disuccinimidyl suberateand identified the cross-linked lysine residues by MS (16–18).Among the identified cross-links, one intersubunit interaction in-volved Rpn11: Lys49 located in the coiled-coil region of Rpt3 is inproximity to Lys281 in the C-terminal domain of Rpn11 (Table 1).Previous tandem affinity purification experiments revealed Rpt3/Rpt6/Rpn8/Rpn11 forming a subcomplex (30), consistent withthe proximity of Rpt3 and Rpn11. The placement of Rpn11 closeto the AAA-ATPases is not unexpected: The deubiquitylating ac-tivity of Rpn11 is ATP dependent, but Rpn11 is not an ATPaseitself, which implies cooperativity of substrate translocation anddeubiquitylation (13, 14). Thus, the cross-linking data suggestthat part of the Rpn11 C-terminal domain is accessible frominside the cavity surrounding the AAA-ATPase, whereas theC terminus itself projects to the periphery of the RP. There are29 residues between Lys281 and the C terminus, which are suffi-cient to bridge the ∼30-Å space between the proteasome mouthand the periphery. Interestingly, it is exactly these ∼30 C-terminal

Fig. 1. Structure of the 26S proteasome from S. pombe. Model of the 26S proteasome and two different views rotated around the pseudo-sevenfold axis ofthe CP by 90°. The isosurface threshold was set to include a protein mass of 755 kDa for the CP. Secondary structure elements like bihelical repeats can be clearlydiscerned (A, *). B shows the isosurface representations of the model (lower half). The transparent view through the reconstruction shows the fitted atomicmodels of the CP (red, **: α-helix of subunit α4) and the AAA-ATPase (blue). A mesh representation of the reconstruction with an overlay isosurface in greenhighlighting the main variances among the particles is shown in C.

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residues that have been shown to be essential for the role ofRpn11 in mitochondrial biogenesis (31).

Based on the Ab-labeling and the cross-linking data, we canapproximately map the C-terminal domain of Rpn11. Fromthe cross-link Rpt3:Lys49-Rpn11:Lys281, we conclude that thesetwo lysine residues are ∼20 Å apart from each other; the C-term-inal domain of Rpn11 is placed at the intersection of the spherecentered on the FLAG-Ab and a sphere around Lys49 of Rpt3(Fig. 3C). At this point, we cannot precisely map the deubiqui-tylating site, which is located in the N-terminal MPN (Mpr1/Pad1 N-terminal) domain. However, it appears reasonable thatit is accessible from the “pharynx” between base and lid, such thatthe placement of Rpn11 near the mouth of the AAA-ATPase isideally positioned for Ub removal from substrates immediatelyprior to substrate translocation into the CP.

Quaternary Structure of the CP–AAA-ATPase Subcomplex. We pre-viously suggested a model for the quaternary structure of theAAA-ATPase hexamer and its position on the CP based onthe cryo-EM map of the D. melanogaster 26S proteasome andprotein–protein interactions from the literature (22). The pre-dicted AAA-ATPase topology, Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5was recently confirmed by disulfide engineering (32). Our sug-gested positioning of the AAA-ATPase hexamer is however notin accordance with refs. 33 and 34: From interactions of the iso-lated C-terminal peptides of Rpt2 and Rpt5 with the CP subunits,a different CP-AAA-ATPase topology was suggested, which is notcompatible with our model and the protein–protein interactionsunderlying it.

The cross-linking and MS data revealed several interactionswithin the AAA-ATPase and between CP and the AAA-ATPasesubunits (Table 1). We could identify four different cross-linksbetween Rpt and CP subunits: two different residue pairs linkRpt1-α4 and Rpt6-α2, respectively (Fig. 4A). All four cross-linksare in excellent agreement with our model. In fact, no otherAAA-ATPase rotation (Fig. 4B) can fulfill these restraints. Thus,our cross-linking data derived from the fully assembled 26S pro-

Fig. 2. Classification of S. pombe 26S proteasomes reveals variations inthe RP. Five class averages obtained by focused classification (CP in red,AAA-ATPase-ring in blue, remaining RP in brown) are shown in A. Major dif-ferences of each class to the overall average correlate to the localization ofthe variance map shown in Fig. 1 (B) Additional density in green; (C) missingdensity in yellow. As expected from ML3D classification, a subset of 26S pro-teasomes is lacking the extra mass (class 4). Further classes show additionaldensities in the region between base and lid, close to the upper AAA-ATPasering (classes 2, 3, and 5).

Fig. 3. Monoclonal anti-FLAG antibodies bind to the Rpn11–C-terminal3xFLAG-tag in intact 26S proteasomes. After incubation of proteasomes withanti-FLAG-antibody, extra densities are clearly visible in 2D class averages(A, Top Left: control). After classification and 3D reconstruction (B) the centerof mass of the antibody can be clearly seen on the top of the RP lid (green).The cross-link between Lys49 in the coiled-coil region of Rpt3 (C, yellow) andLys281 of Rpn11 has an estimated length of 20 Å and allows mapping Rpn11:Lys281 within a sphere of 20-Å radius (yellow sphere). A sphere of ∼80-Å ra-dius (dotted line) with its central point in the center of mass of the antibodydepicts the possible localization of the IgG bindings site.

Table 1. Crosslinked lysines in the RP of S.pombe proteasomes

Residue 1 Residue 2 Cα-Cα, Å

Intra-ATPaseRpt4:71 Rpt5:124 15Rpt4:318 Rpt5:217 9Rpt5:77 Rpt4:38 15Rpt3:299 Rpt6:357 16Rpt4:71 Rpt5:124 15Rpt3:186 Rpt4:164 24Rpt5:72 Rpt4:41 13Rpt4:37 Rpt4:41 6Rpt3:242 Rpt3:299 17Rpt5:72 Rpt4:38 10Intra-ATPase cavityRpt5:124 Rpt3:207 34Rpt5:124 Rpt4:205 31Rpt6:84 Rpt6:218 33Rpt1:120 Rpt4:205 35Rpt1:124 Rpt3:207 39ATPase—20Salpha4:168 Rpt1:422 17alpha4:168 Rpt1:426 22alpha2:176 Rpt6:390 17alpha2:176 Rpt6:347 16ATPase—lidRpt3:49 Rpn11:281 NA

Identified cross-linked lysines involving the AAA-ATPase hexamer. Thedata were classified according to the positions of the cross-linkedresidues. For each cross-link, both identified lysine residues and theirdistance in our CP-ATPase model are shown. The intra-ATPase cross-links are consistent with the experimentally confirmed Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5 topology.

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teasome comply with the published protein–protein interactiondata, such as Rpt2 binding to α4 (35, 36), as well as genetic datasuggesting Rpt2-α3 interaction (37), but they do conflict with theinteractions reported for the synthetic C-terminal Rpt peptides(e.g., Rpt5-α3 and Rpt5-α4) (33, 34). This discrepancy mightbe explained by different binding specificities of the full-lengthproteins as compared to the short C-terminal peptides.

Cross-Linking Data Suggest a Similar Translocation Mechanism as inHslU. We identified five cross-links connecting residues locatedin the AAA-ATPase channel. All of these cross-links connectedresidues in the proximal ring of the N-terminal domains and thehighly conserved Ar-Φ loop located in the AAA ring (Fig. 4A).The distances of these residues in our AAA-ATPase model(∼35 Å) exceeded the distance that is typically bridged by disuc-cinimidyl suberate (<20 Å). Indeed, it has been suggested thatPAN and the proteasomal AAA-ATPases possess a translocationmechanism similar to that of heat shock locus U (HslU) (38). InHslU, the Ar-Φ loop undergoes substantial conformationalchanges upon ATP hydrolysis, which is believed to enable it topull the substrate through the pore (28). The identified cross-links suggest that the Ar-Φ loop of the AAA-ATPase of the 26Sproteasome also displays a structural flexibility similar to thatobserved with HslU.

ConclusionsThe 26S proteasome from S. pombe is constituted of the canoni-cal subunits α1–7, β1–7, Rpt1–6, Rpn1–9, and Rpn11–12,whereas the subunits Rpn10, Rpn13a/b, Uch2, and Ubp6 are pre-sent in significant, yet varying, amounts. InD. melanogaster and inS. pombe, the occupancy of the extra mass derived from the classaverages correlated to the abundance of Rpn10 as determined byMS. Three-dimensional reconstruction of vitrified proteasomesyields an EM map with a resolution of 9.1 Å. Patches with thecharacteristic features of short α-helical repeats become discern-ible, supporting the prediction for large portions of many RPsubunits to adopt α-solenoidal folds. Ab labeling and the cross-linking data suggest that the C-terminal domain of Rpn11 isplaced near the opening of the AAA-ATPase cavity, allowing ef-ficient access to its substrate and release of Ub. At the base–lidinterface, a belt of high variance surrounds the AAA-ATPase.The distal part of the lid complex, however, is rather invariable,thus providing a rigid frame or roof. Classification of particlesaccording to the belt of major variance is indicative of the rever-sible binding of PIPs, Ub, or substrate. With the availability of

much larger datasets than used in this study, allowing an exhaus-tive classification and/or the use of different biochemically de-fined proteasome-substrate states, it will be possible to analyzethis belt “high activity” in more detail.

Materials and MethodsProtein Purification and Analysis. Purification of intact 26S proteasomescontaining a 3xFLAG-tag at the C terminus of Rpn11 were purified fromS. pombe (972 h+ rpn11∷RPN11-3xFLAG-His3) essentially as described (39)(SI Text). In short, cleared cell lysates were incubated with anti-FLAG M2agarose beads (Sigma), washed, and the bound proteins eluted with 3xFLAGpeptide (Sigma). The elute was applied to a sucrose gradient and fractionswere tested for protease activity. The fraction from sucrose gradients withthe highest proteolytic activity was analyzed by MS as described (9), usedfor cryo-EM and chemical cross-linking (SI Text). In brief, purified protea-somes were concentrated, cross-linked with disuccinimidyl suberate d0/d12(Creativemolecules, Inc.), and trypsin digested (Promega). Liquid chromato-graphy-MS/MS analysis was carried out on an LTQ Orbitrap XL mass spectro-meter (Thermo Electron). Data were searched using xQuest (40) and furtheranalyzed manually. For Ab labeling, purified proteasomes were incubatedwith M2 Anti-FLAG Ab (Sigma) for 1 h at 4 °C.

EM. Focal pairs with a nominal defocus of 1–3 μm were recorded on anEagle CCD camera using an FEI Tecnai F20 microscope at 200 kV (finalmagnification, 63,500×; object pixel size, 2.2 Å) in a semiautomated mannerusing the SerialEM software package (41). The contrast transfer function wasdetermined and micrographs were deconvoluted by phase flipping and com-pensation for the modulation transfer function of the CCD camera (42). From∼21;000 micrographs, ∼320;000 particles were selected for 3D reconstruction(26S ∼270;000, 26S-Ab ∼50;000).

Reconstruction, 3D Variance, and Classification. The initial 3D model for refine-ment was the D. melanogaster 26S proteasome density filtered to 4 nm. Datawere sorted using ML3D classification (Fig. S3) (21) and densities were furtherrefined as described in ref. 43. The temperature factor was determined (44)and applied to the final reconstruction (∼ − 250 Å2). The variance map wascalculated according to ref. 27. For focused classification, angularly refinedparticles were C2 symmetrized, masked, and split into 30 iteratively opti-mized groups (Fig. S7). The position and radius of the mask was determinedby evaluating the variance map.

Modeling. Comparative models of the S. pombe 20S proteasome and theAAA-ATPases were built as described previously (22). The positions of CPand AAA-ATPase hexamer were locally refined in the experimental mapusing the University of California, San Francisco Chimera (45).

ACKNOWLEDGMENTS. We thank Y. Saeki (Tokyo Metropolitan Institute ofMedical Science) for providing a plasmid encoding the 3xFLAG-His-Tag

Fig. 4. Structural and mechanistic depiction of the regulatory particle. Rpt1-α4 and Rpt6-α2 cross-links (A, yellow) between AAA-ATPase (blue) and CP (red)corroborate the previously suggested CP-AAA topology (32). The cross-links between AAA-ATPase subunits (A, green) confirm the Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5 topology of the AAA-ATPase hexamer. Cross-links in the AAA cavity are indicative of inducible conformational changes of central rings (A, magenta).(B) Top-view visualizing the positioning of the AAA-ATPase ring (t1–t6, center of AAA ring: white asterisk) relative to the α-ring of the CP (α1–α7, center: blackasterisk). (C) Integrated model of the RP and α-ring of the CP: Ubiquitylated substrates are recognized at the RP (e.g., Rpn10 or Rpn13) of the 26S proteasome,deubiquitylated (e.g., Rpn11 or Uch2) for Ub recycling and translocated (Rpt1–6) through the AAA-ATPase cavity and the opened gate (Rpt2, Rpt5) of the CP fordegradation (extra mass, cyan; Rpt1–6, as in B; Rpt3–Rpn11 cross-link, as in Fig. 3).

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and M. Shravan for providing S. pombe support. E.S. is a recipient of a JapanSociety for the Promotion of Science Research Fellowship for YoungScientists. F.F. thanks Human Frontier Science Project Organization for a ca-

reer development award. This work was supported in part by funding fromthe European Union Seventh Framework Program PROSPECTS (ProteomicsSpecification in Space and Time Grant HEALTH-F4-2008-201648).

1. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway:Destruction for the sake of construction. Physiol Rev 82:373–428.

2. Hershko A, Ciechanover A (1998) The ubiquitin system.Annu Rev Biochem 67:425–479.3. Voges D, Zwickl P, Baumeister W (1999) The 26S proteasome: A molecular machine

designed for controlled proteolysis. Annu Rev Biochem 68:1015–1068.4. Hershko A, Ciechanover A, Varshavsky A (2000) Basic medical research award. The

ubiquitin system. Nat Med 6:1073–1081.5. Baumeister W, Walz J, Zuhl F, Seemuller E (1998) The proteasome: Paradigm of a

self-compartmentalizing protease. Cell 92:367–380.6. Groll M, et al. (1997) Structure of 20S proteasome from yeast at 2.4 A resolution.

Nature 386:463–471.7. Lowe J, et al. (1995) Crystal structure of the 20S proteasome from the archaeon

T. acidophilum at 3.4 A resolution. Science 268:533–539.8. Förster F, Lasker K, Nickell S, Sali A, Baumeister W (2010) Towards an integrated

structural model of the 26S proteasome. Mol Cell Proteomics 9:1666–1677.9. Nickell S, et al. (2009) Insights into the molecular architecture of the 26S proteasome.

Proc Natl Acad Sci USA 106:11943–11947.10. Smith DM, et al. (2007) Docking of the proteasomal ATPases’ carboxyl termini in the

20S proteasome’s alpha ring opens the gate for substrate entry. Mol Cell 27:731–744.11. Djuranovic S, et al. (2009) Structure and activity of the N-terminal substrate recogni-

tion domains in proteasomal ATPases. Mol Cell 34:580–590.12. Zhang F, et al. (2009) Structural insights into the regulatory particle of the proteasome

from Methanocaldococcus jannaschii. Mol Cell 34:473–484.13. Verma R, et al. (2002) Role of Rpn11 metalloprotease in deubiquitination and

degradation by the 26S proteasome. Science 298:611–615.14. Yao T, Cohen RE (2002) A cryptic protease couples deubiquitination and degradation

by the proteasome. Nature 419:403–407.15. Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the

proteasome. Annu Rev Biochem 78:477–513.16. Leitner A, et al. (2010) Probing native protein structures by chemical cross-linking,

mass spectrometry, and bioinformatics. Mol Cell Proteomics 9:1634–1649.17. Maiolica A, et al. (2007) Structural analysis of multiprotein complexes by cross-linking,

mass spectrometry, and database searching. Mol Cell Proteomics 6:2200–2211.18. Seebacher J, et al. (2006) Protein cross-linking analysis using mass spectrometry, iso-

tope-coded cross-linkers, and integrated computational data processing. J ProteomeRes 5:2270–2282.

19. Belote JM, Zhong L (2009) Duplicated proteasome subunit genes in Drosophila andtheir roles in spermatogenesis. Heredity 103:23–31.

20. Isasa M, et al. (2010) Monoubiquitination of RPN10 regulates substrate recruitment tothe proteasome. Mol Cell 38:733–745.

21. Scheres SH, et al. (2007) Disentangling conformational states of macromolecules in3D-EM through likelihood optimization. Nat Methods 4:27–29.

22. Förster F, et al. (2009) An atomic model AAA-ATPase/20S core particle sub-complex ofthe 26S proteasome. Biochem Biophys Res Commun 388:228–233.

23. Kajava AV (2002) What curves alpha-solenoids? Evidence for an alpha-helicaltoroid structure of Rpn1 and Rpn2 proteins of the 26S proteasome. J Biol Chem277:49791–49798.

24. Scheel H, Hofmann K (2005) Prediction of a common structural scaffold for protea-some lid, COP9-signalosome and eIF3 complexes. BMC Bioinf 6:71–81.

25. Enchev RI, Schreiber A, Beuron F, Morris EP (2010) Structural insights into theCOP9 signalosome and its common architecture with the 26S proteasome lid andeIF3. Structure 18:518–527.

26. Ciccarelli FD, Izaurralde E, Bork P (2003) The PAM domain, a multi-protein complex-associated module with an all-alpha-helix fold. BMC Bioinf 4:64–69.

27. Penczek PA, Yang C, Frank J, Spahn CM (2006) Estimation of variance in single-particlereconstruction using the bootstrap technique. J Struct Biol 154:168–183.

28. Varshavsky A (2006) The early history of the ubiquitin field. Protein Sci 15:647–654.29. WangQ, Young P,Walters KJ (2005) Structure of S5a bound tomonoubiquitin provides

a model for polyubiquitin recognition. J Mol Biol 348:727–739.30. Gavin AC, et al. (2006) Proteome survey reveals modularity of the yeast cell machinery.

Nature 440:631–636.31. Rinaldi T, et al. (2008) Dissection of the carboxyl-terminal domain of the proteasomal

subunit Rpn11 in maintenance of mitochondrial structure and function. Mol Biol Cell19:1022–1031.

32. Tomko RJ, Jr, Funakoshi M, Schneider K, Wang J, Hochstrasser M (2010) Heterohexa-meric ring arrangement of the eukaryotic proteasomal ATPases: Implications forproteasome structure and assembly. Mol Cell 38:393–403.

33. Yu Y, et al. (2009) Interactions of PAN’s C-termini with archaeal 20S proteasome andimplications for the eukaryotic proteasome-ATPase interactions. EMBO J 29:692–702.

34. Gillette TG, Kumar B, Thompson D, Slaughter CA, DeMartino GN (2008) Differentialroles of the COOH termini of AAA subunits of PA700 (19S regulator) in asymmetricassembly and activation of the 26S proteasome. J Biol Chem 283:31813–31822.

35. Zhang Z, et al. (2000) Structural and functional characterization of interactionbetween hepatitis B virus X protein and the proteasome complex. J Biol Chem275:15157–15165.

36. Chen C, et al. (2008) Subunit-subunit interactions in the human 26S proteasome.Proteomics 8:508–520.

37. Bajorek M, Glickman MH (2004) Keepers at the final gates: Regulatory complexes andgating of the proteasome channel. Cell Mol Life Sci 61:1579–1588.

38. Zhang F, et al. (2009) Mechanism of substrate unfolding and translocation by theregulatory particle of the proteasome from Methanocaldococcus jannaschii. Mol Cell34:485–496.

39. Saeki Y, Isono E, Toh EA (2005) Preparation of ubiquitinated substrates by the PYmotif-insertion method for monitoring 26S proteasome activity. Methods Enzymol399:215–227.

40. Rinner O, et al. (2008) Identification of cross-linked peptides from large sequencedatabases. Nat Methods 5:315–318.

41. Mastronarde DN (2005) Automated electron microscope tomography using robustprediction of specimen movements. J Struct Biol 152:36–51.

42. Nickell S, et al. (2005) TOM toolbox acquisition and analysis for electron tomography.J Struct Biol 149:227–234.

43. Scheres SH, Nunez-Ramirez R, Sorzano CO, Carazo JM, Marabini R (2008) Imageprocessing for electron microscopy single-particle analysis using XMIPP. Nat Protoc3:977–990.

44. Rosenthal PB, Henderson R (2003) Optimal determination of particle orientation,absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol333:721–745.

45. Pettersen EF, et al. (2004) UCSF Chimera—a visualization system for exploratoryresearch and analysis. J Comput Chem 25:1605–1612.

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