Supporting InformationBohn et al. 10.1073/pnas.1015530107SI Materials and MethodsProtein Purification and Analysis. Intact 26S proteasome containinga 3xFLAG-tag at the C terminus of Rpn11 were purified fromSchizosaccharomyces pombe (972 h+ rpn11∷RPN11-3xFLAG-His3) essentially as described (1). Cells were cultured in yeast ex-tract liquid medium at 32°, and subsequently lysed in lysis buffer(50 mM Tris •HCl, pH 7.1, 10 mM NaCl, 10 mM MgCl2, 4 mMATP, 5 mMDTT, 10 mM creatine phosphate, 3 units per millilitercreatine phosphate kinase, 10% glycerol). Lysates were clearedby centrifugation and incubated with anti-FLAG M2 agarosebeads (Sigma) for 2 h at 4 °C, which were then washed threetimes. The bound proteins were eluted with 400 g∕mL 3xFLAGpeptide (Sigma) and subjected to a 15–40% sucrose gradient.Each fraction was tested for protease activity using a succinyl-leucine-leucine-valine-tyrosine-7-amido-4-methylcoumarin assayand analyzed by negative stain EM to determine the approximateratio of intact 26S. The fraction with the highest proteolytic ac-tivity showed the highest 26S holocomplex concentration and wasanalyzed by MS as described (2), used for chemical cross-linkingand cryo-EM. For antibody (Ab) labeling, purified proteasomeswere incubated with M2 Anti-FLAG Ab (Sigma) for 1 h at 4 °Cprior to vitrification.

Chemical Cross-Linking. Purified proteasome samples were concen-trated using an Roti-Spin-MINI-30 cutoff filter device (Roth) to1 mg∕mL. Fifty microliter samples were cross-linked with 1 mMdisuccinimidyl suberate d0/d12 (Creativemolecules, Inc.) in20 mM Hepes, pH 7.1, 20 mM NaCl, 10 mM MgCl2, 4 mM

ATP, 5 mM DTT, 10 mM creatine phosphate, and 3 units permilliliter creatine phosphate kinase at 35 °C for 30 min. The re-action was quenched by adding 50 mM ammonium bicarbonatefor 10 min at 35 °C.

The protein samples were reduced with 1 mM Tris (2-carbox-yethyl) phosphine hydrochloride (Pierce) at 37 °C for 30 min andsubsequently alkylated with 2 mM iodoacetamide (Sigma-Al-drich) for 10 min at room temperature in the dark. For digestion10% acetonitrile, urea (1 M final concentration) and 2% wt∕wttrypsin (Promega) was added. Digestion was carried out at 37 °Covernight and stopped by acidification to 1% ðwt∕volÞ trifluoroa-cetic acid. Peptides were purified with C18 MicroSpin columns(The Nest Group), according to the manufacturer’s protocol.Subsequently cross-linked peptides were enriched using a peptidesize-exclusion chromatography (manuscript in preparation).Liquid chromatography-MS/MS analysis was carried out on anLTQ Orbitrap XL mass spectrometer (Thermo Electron).

Data were searched using xQuest (3) in iontag mode against adatabase containing the protein sequences of previously identi-fied proteasome proteins with a precursor mass tolerance of10 ppm. For matching of fragment ions, tolerances of 0.2 Dafor common ions and 0.3 Da for cross-link ions were used.Cross-linked peptides identified with a linear discriminantscore > 25, corresponding to a false discovery rate <5% (4), werefurther analyzed manually. Thereby, matching ion series on bothcross-linked peptide chains were reviewed by visual inspection inorder to match the most abundant peaks.

1. Saeki Y, Isono E, Toh EA (2005) Preparation of ubiquitinated substrates by the PY

motif-insertion method for monitoring 26S proteasome activity. Methods Enzymol

399:215–227.

2. Nickell S, et al. (2009) Insights into the molecular architecture of the 26S proteasome.

Proc Natl Acad Sci USA 106:11943–11947.

3. Rinner O, et al. (2008) Identification of cross-linked peptides from large sequence

databases. Nat Methods 5:315–318.

4. Leitner A, et al. (2010) Probing native protein structures by chemical cross-linking,mass spectrometry, and bioinformatics. Mol Cell Proteomics 9:1634–1649.

5. Penczek PA, Frank J, Spahn CM (2006) A method of focused classification, based onthe bootstrap 3D variance analysis, and its application to EF-G-dependent transloca-tion. J Struct Biol 154:184–194.

6. 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.

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Fig. S1. Subunit composition of 26S proteasomes from S. pombe compared to Drosophila melanogaster. Sucrose gradient fraction showing highest activity insuccinyl-leucine-leucine-valine-tyrosine-7-amido-4-methylcoumarin cleavage was trypsin digested and analyzed by MS. Peptide intensities were quantifiedfour times independently, normalized (2), and compared to data from D. melanogaster (2). In S. pombe, the canonical 26S proteasome subunits α1–7,β1–7, Rpt1–6, Rpn1–3, and Rpn5–12 are approximately present in an equimolar ratio. Rpn13 and the proteasome interacting proteins (PIPs) Uch2 andUbp6 were found in substoichiometric amounts. Rpn15/Sem1p was detected by a single peptide, whereas for all other subunits 23� 11 peptides have beendetected; thus, the determined Rpn15 stoichiometry is inaccurate. Notably, only the amounts of Rpn10, Rpn13, and Uch2/Uch37 differ significantly between S.pombe and D. melanogaster 26S proteasomes. Two orthologs of Rpn13 have been detected in S. pombe (* left arrow Rpn13b, right arrow Rpn13a). CP, coreparticle; RP, regulatory particle.

Fig. S2. Comparison of unlabeled and Ab-labeled 26S proteasome particles. Zoom into typical electron micrographs of S. pombe 26S proteasomes without (A)or with (B) Ab incubation prior to vitrification shows single (A, box) and “twin” (B, box) particles. Twins—two 26S proteasomes displaying four identical 3xFLAGepitopes linked by two anti-FLAG-Abs—typically show only one orientation when embedded in ice. (C) Two-dimensional average of ∼600 twins. The obtainedclass average can be used to model the relative orientation of two 3D density maps of the 26S proteasome (D).

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Fig. S3. Classification of 26S proteasomes from S. pombe. From the initial ∼20;000 micrographs, a total number of ∼270;000 particles have been selected. Theinitial reconstruction yielded a density map at 9.1 Å (class A). To sort out assembly intermediates, we performed the maximum likelihood-based classificationmethod (ML3D). Particles were classified into two groups (class B and C), of which one showed all the features of 26S holocomplexes, whereas the other clearlycorresponded to partially (dis)assembled 26S proteasomes. The reconstruction of fully assembled proteasomes (class B2) yielded a map of 9.1-Å resolution. In asecondML3D classification step, holocomplexes separated according to their difference of the distinct “extra mass” in the cap region (class D1 with one and D2

with two extra masses). Using classification focused on the area with the highest variance (Figs. S8–S7), particles belonging to class B were further classified into30 classes (colored classes are enlarged in Fig. 2).

Fig. S4. Model comparison of S. pombe and D. melanogaster 26S proteasomes. The map of D. melanogaster with a nominal resolution of 20 Å (2) (Left) wascompared to the model of S. pombe, bandpass filtered to the same resolution (Right). The isosurface levels of both models were chosen such that they en-capsulate the volume expected for the core particle. At this resolution, both reconstructions are very similar. However, a clear difference in the occupancy of theextra mass can be seen (blue arrows). A further density difference at the base of the regulatory particle (“hinge” region, red arrows) is located in an area that ishighly variable in both S. pombe and D. melanogaster 26S proteasomes.

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Fig. S5. Fourier shell correlation (FSC) curves of reconstructed models. Classes A, B, and D2 were refined with a final angular increment of 0.2° (data split into39 contrast transfer function groups), without imposed symmetry (A, B1) or with imposed C2 symmetry (B2, D2). Imposed C2 symmetry improved the resolutionto 9.1 and 6.7 Å at FSC of 0.5 and 0.3, respectively. When not explicitly noted, the FSC criterion of 0.5 is used.

Fig. S6. Bihelical repeats in the regulatory particle density. In the 26S proteasome map (A) patches with apparent helical segments are discernible (rectangle;enlarged in B). The density of tetratricopeptide repeats from Sec17 (Protein Data Bank ID code 1QQE) filtered to 9.7-Å resolution (C) resembles these patches.(D) Ribbon representation of 1QQE.

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Fig. S7. Simplified example of focused classification with two classes. To separate the single particles according to their conformational states, we grouped theparticles into N classes according to their structural differences within a sphere “m” positioned at an area of interest in the 3D map. In our case, the mask waspositioned at the mouth region of the regulatory particle (RP) (Fig. S9). We chose an algorithm, which is similar to the focused classification suggested by (5).Our procedure differs in the algorithm for assignment of the conformational classes. The first step of our classification procedure was to determine the 3Dstructure from the whole dataset by projection matching using Xmipp (6). For subsequent classification of particles according to conformations, each particlekeeps this angular assignment. We simultaneously classified both RPs capping the core particle by including each particle twice into the analyzed dataset: onecopy with the original projection angles and translation vectors and one copy with the C2 transformed rotation angles. Aim of the classification process was todivide the dataset into N different classes, such that their correlation to the corresponding 3D model is maximized. For optimization of the particle assign-ments, we chose the following expectation maximization approach: To initialize the actual classification particles were randomly split into N equally populatedsubsets, the corresponding N 3D models were reconstructed (“seeds”), and projections of these N models were calculated (A). The projections as well as allindividual particles were multiplied with a projection of the 3D mask (alignment parameters from projection matching) (B). Within every angular class, eachparticle was correlated to the corresponding N model projections (C). Each particle was assigned to the model with the highest correlation value. Again, Nmodels were calculated using this new particle assignment (D). The process was iterated until the number of particles changing their class was below 1%.

Fig. S8. Comparison of classification methods. Two-dimensional class averages (A), derived from Principle Component Analysis and K-means clustering, arequalitatively similar to projections of classes of the focused classification approach. (B) Three-dimensional classes selected from Fig. S9. (C) Respective back-projection of models shown in B. D shows the back-projection of the 3D average of all particles as well as the variance among them (arrows pointing to the“mouth” region of the regulatory particle, between lid and base).

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Fig. S9. Classes of S. pombe 26S proteasomes. From a dataset of ∼185;000 26S holocomplexes, 30 classes is themaximum number which provides an amount ofparticles per model (∼4;000–15;000) yielding reconstructions of sufficient resolution (3–4 nm). The classification of the regulatory particle resulted in modelswith varying densities in the region surrounding the AAA-ATPase, between lid, base, and extra mass (boxed classes were chosen for visualization in Fig. 2). Themask used for focused classification is shown in A. The occupation of each class is depicted in the diagram and respective numbers of particles per class aregiven.

Movie S1. The 26S proteasome rotated around its pseudo-sevenfold symmetry axis. The movie shows the C2-model of the 26S proteasome (class B, Fig. S3)from S. pombe, rotating 360° around its pseudo-sevenfold symmetry axis. The resolution of the reconstruction was determined to be 9.1 and 6.7 Å at FSC of 0.5and 0.3.

Movie S1 (AVI)

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Movie S2. Movie of the variations in the 19S regulatory particle (RP). The movie shows three cycles of 30 classes (Fig. S9), derived by focused classification(Fig. S7). The first class was randomly selected. The class for the second frame was the class with the highest cross-correlation coefficient among the remaining29 classes when compared to the first class, and so forth. The remaining densities in the lid of the RP and the core particle (only partially displayed) show almostno variation when compared to the region between lid and base of the shown RP.

Movie S2 (AVI)

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