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- EMDB-9042: Yeast 26S proteasome bound to ubiquitinated substrate (1D* motor ... -

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Entry
Database: EMDB / ID: 9042
TitleYeast 26S proteasome bound to ubiquitinated substrate (1D* motor state)
Map dataYeast 26S proteasome bound to ubiquitinated substrate (1D* motor state)
SampleSubstrate-engaged 26S proteasome in the 1D* state
  • proteasome
  • substrate
  • (Proteasome subunit alpha type- ...) x 6
  • Probable proteasome subunit alpha type-7
  • (26S proteasome regulatory subunit ...) x 5
  • 26S proteasome subunit RPT4
  • model substrate polypeptide
  • (ligand) x 2
Function / homologyNucleophile aminohydrolases, N-terminal / Proteasome beta-type subunit, conserved site / Proteasome subunit / ATPase family associated with various cellular activities (AAA) / Proteasome subunit alpha 7-like / Proteasome subunit alpha 3 / Proteasome alpha-subunit, N-terminal domain / Proteasome, subunit alpha/beta / AAA+ ATPase domain / ATPase, AAA-type, core ...Nucleophile aminohydrolases, N-terminal / Proteasome beta-type subunit, conserved site / Proteasome subunit / ATPase family associated with various cellular activities (AAA) / Proteasome subunit alpha 7-like / Proteasome subunit alpha 3 / Proteasome alpha-subunit, N-terminal domain / Proteasome, subunit alpha/beta / AAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / 26S proteasome regulatory subunit P45-like / Proteasome alpha-type subunit / 26S Proteasome regulatory subunit 6B / P-loop containing nucleoside triphosphate hydrolase / 26S proteasome regulatory subunit 8 / Proteasomal ATPase OB C-terminal domain / Proteasome subunit alpha5 / Proteasome subunit alpha6 / Proteasome subunit alpha2 / Proteasome subunit alpha4 / Proteasome subunit alpha 1 / 26S Proteasome regulatory subunit 4 / 26S Proteasome regulatory subunit 7 / Proteasome subunit A N-terminal signature / Proteasomal ATPase OB C-terminal domain / 26S Proteasome regulatory subunit 6A / SCF-beta-TrCP mediated degradation of Emi1 / Antigen processing: Ubiquitination & Proteasome degradation / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / CDK-mediated phosphorylation and removal of Cdc6 / Orc1 removal from chromatin / Neutrophil degranulation / Ub-specific processing proteases / MAPK6/MAPK4 signaling / TNFR2 non-canonical NF-kB pathway / AUF1 (hnRNP D0) binds and destabilizes mRNA / ABC-family proteins mediated transport / SCF(Skp2)-mediated degradation of p27/p21 / Cross-presentation of soluble exogenous antigens (endosomes) / Proteasome alpha-type subunit profile. / Proteasome alpha-type subunits signature. / AAA-protein family signature. / proteasome regulatory particle assembly / nonfunctional rRNA decay / nuclear proteasome complex / proteasome-activating ATPase activity / proteasome regulatory particle, base subcomplex / cellular protein-containing complex localization / cytosolic proteasome complex / peptide catabolic process / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / proteasome storage granule / proteasome core complex / proteasomal ubiquitin-independent protein catabolic process / proteasome endopeptidase complex / ubiquitin-dependent ERAD pathway / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / TBP-class protein binding / positive regulation of transcription elongation from RNA polymerase II promoter / proteasomal protein catabolic process / nucleotide-excision repair / positive regulation of RNA polymerase II transcriptional preinitiation complex assembly / positive regulation of protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of DNA-binding transcription factor activity / ubiquitin-dependent protein catabolic process / chromatin remodeling / positive regulation of DNA-binding transcription factor activity / endopeptidase activity / ATPase activity / mRNA binding / protein domain specific binding / ubiquitin protein ligase binding / mitochondrion / ATP binding / identical protein binding / nucleus / cytoplasm / 26S proteasome regulatory subunit 7 homolog / 26S proteasome subunit RPT4 / 26S proteasome regulatory subunit 4 homolog / Proteasome subunit alpha type-4 / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 6A / Proteasome subunit alpha type-2 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-1 / Probable proteasome subunit alpha type-7 / 26S proteasome regulatory subunit 8 homolog
Function and homology information
SourceSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 4.43 Å resolution
Authorsde la Pena AH / Goodall EA / Gates SN / Lander GC / Martin A
CitationJournal: Science / Year: 2018
Title: Substrate-engaged 26 proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.
Authors: Andres H de la Peña / Ellen A Goodall / Stephanie N Gates / Gabriel C Lander / Andreas Martin
Abstract: The 26 proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the ...The 26 proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26 proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.
Validation ReportPDB-ID: 6ef0

SummaryFull reportAbout validation report
DateDeposition: Aug 15, 2018 / Header (metadata) release: Oct 17, 2018 / Map release: Oct 17, 2018 / Last update: Oct 17, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
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  • Surface view with fitted model
  • Atomic models: : PDB-6ef0
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6ef0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9042.map.gz (map file in CCP4 format, 157217 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
340 pix
1.03 Å/pix.
= 350.2 Å
340 pix
1.03 Å/pix.
= 350.2 Å
340 pix
1.03 Å/pix.
= 350.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density
Contour Level:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum0.0 - 0.19451101
Average (Standard dev.)0.0012396917 (0.006442988)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions340340340
Origin0.00.00.0
Limit339.0339.0339.0
Spacing340340340
CellA=B=C: 350.19998 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z350.200350.200350.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean0.0000.1950.001

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Supplemental data

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Mask #1

Fileemd_9042_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Mask #2

Fileemd_9042_msk_2.map
Projections & Slices
AxesZYX
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Slices (1/2)
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Mask #3

Fileemd_9042_msk_3.map
Projections & Slices
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Slices (1/2)
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Sample components

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Entire Substrate-engaged 26S proteasome in the 1D* state

EntireName: Substrate-engaged 26S proteasome in the 1D* state
Details: Yeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP
Number of components: 19

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Component #1: protein, Substrate-engaged 26S proteasome in the 1D* state

ProteinName: Substrate-engaged 26S proteasome in the 1D* state
Details: Yeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP
Recombinant expression: No

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Component #2: protein, proteasome

ProteinName: proteasome / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast) / Strain: ATCC 204508 / S288c

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Component #3: protein, substrate

ProteinName: substrate / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria) / Strain: BL21

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Component #4: protein, Proteasome subunit alpha type-1

ProteinName: Proteasome subunit alpha type-1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.801549 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #5: protein, Proteasome subunit alpha type-2

ProteinName: Proteasome subunit alpha type-2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.191828 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #6: protein, Proteasome subunit alpha type-3

ProteinName: Proteasome subunit alpha type-3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.080506 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #7: protein, Proteasome subunit alpha type-4

ProteinName: Proteasome subunit alpha type-4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.993475 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #8: protein, Proteasome subunit alpha type-5

ProteinName: Proteasome subunit alpha type-5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.444869 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #9: protein, Proteasome subunit alpha type-6

ProteinName: Proteasome subunit alpha type-6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.634 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #10: protein, Probable proteasome subunit alpha type-7

ProteinName: Probable proteasome subunit alpha type-7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 27.092719 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #11: protein, 26S proteasome regulatory subunit 7 homolog

ProteinName: 26S proteasome regulatory subunit 7 homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.597023 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #12: protein, 26S proteasome regulatory subunit 4 homolog

ProteinName: 26S proteasome regulatory subunit 4 homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.109572 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #13: protein, 26S proteasome regulatory subunit 8 homolog

ProteinName: 26S proteasome regulatory subunit 8 homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.522594 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #14: protein, 26S proteasome regulatory subunit 6B homolog

ProteinName: 26S proteasome regulatory subunit 6B homolog / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.387693 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #15: protein, 26S proteasome subunit RPT4

ProteinName: 26S proteasome subunit RPT4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.386865 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #16: protein, 26S proteasome regulatory subunit 6A

ProteinName: 26S proteasome regulatory subunit 6A / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.497504 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #17: protein, model substrate polypeptide

ProteinName: model substrate polypeptide / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 1.130277 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #18: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Component #19: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 25 mg/ml / pH: 7.6
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 90 %
Details: specimens were manually blotted with Whatman #1 filter paper.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: images were acquired in nanoprobe mode
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1000.0 - -2500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 11656 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 34701 / Details: Camera was operated in counting mode
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION
CTF correction: CTF correction was performed by Relion during reconstruction
Resolution: 4.43 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 5MPC
Output model

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