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- EMDB-21700: SD-like state of human 26S proteasome in complex with non-cleavab... -

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Basic information

Entry
Database: EMDB / ID: EMD-21700
TitleSD-like state of human 26S proteasome in complex with non-cleavable M1-linked hexaubiquitin
Map data
Sample
  • Complex: Human 26S Proteasome with non-cleavable M1-linked hexaubiquitin
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.47 Å
AuthorsChen X / Walters KJ
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)1 ZIA BC011490 United States
National Science Foundation (NSF, United States)1531991 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CCR FLEX program United States
CitationJournal: Structure / Year: 2020
Title: Cryo-EM Reveals Unanchored M1-Ubiquitin Chain Binding at hRpn11 of the 26S Proteasome.
Authors: Xiang Chen / Zachary Dorris / Dan Shi / Rick K Huang / Htet Khant / Tara Fox / Natalia de Val / Dewight Williams / Ping Zhang / Kylie J Walters /
Abstract: The 26S proteasome is specialized for regulated protein degradation and formed by a dynamic regulatory particle (RP) that caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. ...The 26S proteasome is specialized for regulated protein degradation and formed by a dynamic regulatory particle (RP) that caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Its diverse substrates unify as proteasome targets by ubiquitination. We used cryogenic electron microscopy (cryo-EM) to study how human 26S proteasome interacts with M1-linked hexaubiquitin (M1-Ub) unanchored to a substrate and E3 ubiquitin ligase E6AP/UBE3A. Proteasome structures are available with model substrates extending through the RP ATPase ring and substrate-conjugated K63-linked ubiquitin chains present at inhibited deubiquitinating enzyme hRpn11 and the nearby ATPase hRpt4/hRpt5 coiled coil. In this study, we find M1-Ub at the hRpn11 site despite the absence of conjugated substrate, indicating that ubiquitin binding at this location does not require substrate interaction with the RP. Moreover, unanchored M1-Ub binds to this hRpn11 site of the proteasome with the CP gating residues in both the closed and opened conformational states.
History
DepositionApr 14, 2020-
Header (metadata) releaseAug 5, 2020-
Map releaseAug 5, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21700.png

Downloads & links

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Map

FileDownload / File: emd_21700.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.365 Å
Density
Contour LevelBy AUTHOR: 0.005 / Movie #1: 0.005
Minimum - Maximum-0.017471176 - 0.05649843
Average (Standard dev.)0.000103812185 (±0.0013722165)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 614.25 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3651.3651.365
M x/y/z450450450
origin x/y/z0.0000.0000.000
length x/y/z614.250614.250614.250
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS450450450
D min/max/mean-0.0170.0560.000

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Supplemental data

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Sample components

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Entire : Human 26S Proteasome with non-cleavable M1-linked hexaubiquitin

EntireName: Human 26S Proteasome with non-cleavable M1-linked hexaubiquitin
Components
  • Complex: Human 26S Proteasome with non-cleavable M1-linked hexaubiquitin

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Supramolecule #1: Human 26S Proteasome with non-cleavable M1-linked hexaubiquitin

SupramoleculeName: Human 26S Proteasome with non-cleavable M1-linked hexaubiquitin
type: complex / ID: 1 / Parent: 0
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: 7.5
Component:
ConcentrationFormulaName
50.0 mMTris(hydroxymethyl) aminomethane
50.0 mMNaClSodium chloridesodium chloride
1.5 mMATP-gamma-SATP-gamma-S
5.0 mMMgCl2Magnesium chloride
2.0 mMDTTDithiothreitol
10.0 uMZnSO4Zinc sulphate

Details: 50 mM Tris, pH 7.5, 50 mM NaCl, 1.5 mM ATP-gamma-S, 5 mM MgCl2, 2 mM DTT, 10 uM zinc sulfate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 1645 / Average exposure time: 0.2 sec. / Average electron dose: 43.44 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 164645
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.47 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 22600
FSC plot (resolution estimation)

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