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- EMDB-32290: Structure of USP14-bound human 26S proteasome in substrate-engage... -

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Entry
Database: EMDB / ID: EMD-32290
TitleStructure of USP14-bound human 26S proteasome in substrate-engaged state ED2.0_USP14 with the RPN1 density improved
Map dataMap of the human 26S proteasome in state ED2.0_USP14, with the RP/CP low-pass filtered to 4.1/2.8 angstrom and sharpened by a B-factor of -60/0, rescaled to the pixel size of 0.685 angstrom
Sample
  • Complex: 26S proteasome
KeywordsProteasome / AAA-ATPase / Deubiquitinase / USP14 / HYDROLASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZhang S / Zou S / Yin D / Wu Z / Mao Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)11774012 China
CitationJournal: Nature / Year: 2022
Title: USP14-regulated allostery of the human proteasome by time-resolved cryo-EM.
Authors: Shuwen Zhang / Shitao Zou / Deyao Yin / Lihong Zhao / Daniel Finley / Zhaolong Wu / Youdong Mao /
Abstract: Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating ...Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme ubiquitin-specific protease 14 (USP14), which reversibly binds the proteasome and confers the ability to edit and reject substrates. How USP14 is activated and regulates proteasome function remain unknown. Here we present high-resolution cryo-electron microscopy structures of human USP14 in complex with the 26S proteasome in 13 distinct conformational states captured during degradation of polyubiquitylated proteins. Time-resolved cryo-electron microscopy analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14, and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, ubiquitin-dependent activation of USP14 allosterically reprograms the conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide insights into the complete functional cycle of the USP14-regulated proteasome and establish mechanistic foundations for the discovery of USP14-targeted therapies.
History
DepositionNov 25, 2021-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32290.png

Downloads & links

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Map

FileDownload / File: emd_32290.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the human 26S proteasome in state ED2.0_USP14, with the RP/CP low-pass filtered to 4.1/2.8 angstrom and sharpened by a B-factor of -60/0, rescaled to the pixel size of 0.685 angstrom
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.69 Å/pix.
x 640 pix.
= 438.4 Å		0.69 Å/pix.
x 640 pix.
= 438.4 Å		0.69 Å/pix.
x 640 pix.
= 438.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.685 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028
Minimum - Maximum-0.010097492 - 0.087526545
Average (Standard dev.)0.0016315649 (±0.005064258)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 438.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unfiltered, unsharpened map of the human 26S proteasome...

Fileemd_32290_additional_1.map
AnnotationUnfiltered, unsharpened map of the human 26S proteasome in state ED2.0_USP14
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 26S proteasome

EntireName: 26S proteasome
Components
  • Complex: 26S proteasome

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Supramolecule #1: 26S proteasome

SupramoleculeName: 26S proteasome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.5 MDa

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

9216

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53145
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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