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- EMDB-37269: Consensus cryo-EM structure of human 26S proteasomal RP subcomple... -

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Entry
Database: EMDB / ID: EMD-37269
TitleConsensus cryo-EM structure of human 26S proteasomal RP subcomplex (Ea state) bound to K11/K48-branched ubiquitin (Ub) chain composed of three Ub.
Map dataConsensus cryo-EM map of human 26S RP (Ea state) bound to K11/K48-branched ubiquitin (Ub) chain composed of three Ub.
Sample
  • Complex: Human 26S proteasome
    • Complex: 19S regulatory particle (RP) of human 26S proteasome
      • Complex: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome
    • Complex: alpha subunits belonging to the 20S core particle (CP) of human 26S proteasome
    • Complex: K11/K48-branched ubiquitin (Ub) chain composed of three Ub
Keywordsprotein degradation / macromolecular complex / ubiquitin-proteasome system / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHsu STD / Draczkowski P / Wang YS
Funding support Taiwan, 4 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-CDA-109- L08 Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 109-3114-Y-001-001 Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 110-2113-M-001- 050-MY3 Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 110-2311-B-001-013-MY3 Taiwan
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis of K11/K48-branched ubiquitin chain recognition by the human 26S proteasome.
Authors: Piotr Draczkowski / Szu-Ni Chen / Ting Chen / Yong-Sheng Wang / Hsin-An Shih / Jessica Y C Huang / Ming-Chieh Tsai / Shu-Yu Lin / Steven Lin / Rosa Viner / Yuan-Chih Chang / Kuen-Phon Wu / ...Authors: Piotr Draczkowski / Szu-Ni Chen / Ting Chen / Yong-Sheng Wang / Hsin-An Shih / Jessica Y C Huang / Ming-Chieh Tsai / Shu-Yu Lin / Steven Lin / Rosa Viner / Yuan-Chih Chang / Kuen-Phon Wu / Shang-Te Danny Hsu /
Abstract: Beyond the canonical K48-linked homotypic polyubiquitination for proteasome-targeted proteolysis, K11/K48-branched ubiquitin (Ub) chains are involved in fast-tracking protein turnover during cell ...Beyond the canonical K48-linked homotypic polyubiquitination for proteasome-targeted proteolysis, K11/K48-branched ubiquitin (Ub) chains are involved in fast-tracking protein turnover during cell cycle progression and proteotoxic stress. Here, we report cryo-EM structures of human 26S proteasome in a complex with a K11/K48-branched Ub chain. The structures revealed a multivalent substrate recognition mechanism involving a hitherto unknown K11-linked Ub binding site at the groove formed by RPN2 and RPN10 in addition to the canonical K48-linkage binding site formed by RPN10 and RPT4/5 coiled-coil. Additionally, RPN2 recognizes an alternating K11-K48-linkage through a conserved motif similar to the K48-specific T1 binding site of RPN1. The insights gleaned from these structures explain the molecular mechanism underlying the recognition of the K11/K48-branched Ub as a priority signal in the ubiquitin-mediated proteasomal degradation.
History
DepositionAug 25, 2023-
Header (metadata) releaseAug 28, 2024-
Map releaseAug 28, 2024-
UpdateNov 5, 2025-
Current statusNov 5, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37269.png

Downloads & links

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Map

FileDownload / File: emd_37269.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus cryo-EM map of human 26S RP (Ea state) bound to K11/K48-branched ubiquitin (Ub) chain composed of three Ub.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 400 pix.
= 560. Å		1.4 Å/pix.
x 400 pix.
= 560. Å		1.4 Å/pix.
x 400 pix.
= 560. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.6414645 - 1.8406198
Average (Standard dev.)-0.0023035712 (±0.05503743)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 560.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Consensus cryo-EM half map of human 26S RP...

Fileemd_37269_half_map_1.map
AnnotationConsensus cryo-EM half map of human 26S RP (Ea state) bound to K11/K48-branched ubiquitin (Ub) chain composed of three Ub.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus cryo-EM half map of human 26S RP...

Fileemd_37269_half_map_2.map
AnnotationConsensus cryo-EM half map of human 26S RP (Ea state) bound to K11/K48-branched ubiquitin (Ub) chain composed of three Ub.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human 26S proteasome

EntireName: Human 26S proteasome
Components
  • Complex: Human 26S proteasome
    • Complex: 19S regulatory particle (RP) of human 26S proteasome
      • Complex: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome
    • Complex: alpha subunits belonging to the 20S core particle (CP) of human 26S proteasome
    • Complex: K11/K48-branched ubiquitin (Ub) chain composed of three Ub

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Supramolecule #1: Human 26S proteasome

SupramoleculeName: Human 26S proteasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#26
Details: Images of double-capped (RP-CP-RP) 26S proteasome particles were split in half. After combining with subset of single-capped (RP-CP) complexes the signal in particle images was partially ...Details: Images of double-capped (RP-CP-RP) 26S proteasome particles were split in half. After combining with subset of single-capped (RP-CP) complexes the signal in particle images was partially subtracted leaving only the signal of 19S RP together with alpha subunits of the 20S CP proteasomal subcomplex.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #2: 19S regulatory particle (RP) of human 26S proteasome

SupramoleculeName: 19S regulatory particle (RP) of human 26S proteasome / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #12-#25
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome

SupramoleculeName: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome
type: complex / ID: 3 / Parent: 2 / Macromolecule list: #1-#4, #24-#25
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: alpha subunits belonging to the 20S core particle (CP) of human 2...

SupramoleculeName: alpha subunits belonging to the 20S core particle (CP) of human 26S proteasome
type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5-#11
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: K11/K48-branched ubiquitin (Ub) chain composed of three Ub

SupramoleculeName: K11/K48-branched ubiquitin (Ub) chain composed of three Ub
type: complex / ID: 5 / Parent: 1 / Macromolecule list: #26
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
40.0 mMNaClsodium chloride
5.0 mMMgCl2magnesium chloride
2.0 mMC2H6OS2-Mercaptoethanol
0.5 mMC12H8N21,10-Phenanthroline
2.0 mMC10H16N5O13P3adenosine triphosphate
GridPretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: 20mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: incubation time= 3 s blotting time= 2.5 s.
DetailsThe complex was additionally supplemented with an excess of preformed and SEC-purified RPN13:UCHL5 complex.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 3 / Number real images: 15242 / Average exposure time: 1.8 sec. / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 70000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2695911
CTF correctionSoftware - Name: CTFFIND (ver. 4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: Generated by cryoSPARC using Ab-initio Reconstruction job type.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Software - details: Non-uniform Refinment / Number images used: 54794
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) / Software - details: Non-uniform Refinment / Details: Performed in cryoSPARC using Non-uniform.
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 3.1)
Details: 3D variability analysis (3DVA) followed by Heterogenous refinement using 3DVA cluster volumes as starting references.
FSC plot (resolution estimation)

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