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- EMDB-9044: Yeast 26S proteasome bound to ubiquitinated substrate (5T motor state) -

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Basic information

Entry
Database: EMDB / ID: EMD-9044
TitleYeast 26S proteasome bound to ubiquitinated substrate (5T motor state)
Map dataYeast 26S proteasome bound to ubiquitinated substrate (5T motor state)
Sample
  • Complex: Substrate-engaged 26S proteasome in the 5T state
    • Complex: Proteasome
      • Protein or peptide: x 13 types
    • Complex: substrate
      • Protein or peptide: x 1 types
  • Ligand: x 2 types
Keywords26S Proteasome / ATPase / AAA+ / Protease / Motor protein / Ubiquitin
Function / homology
Function and homology information


proteasome regulatory particle assembly / mitotic cell cycle phase transition / cytosolic proteasome complex / protein-containing complex localization / proteasome-activating activity / proteasome regulatory particle, base subcomplex / cyclin-dependent protein serine/threonine kinase regulator activity / nonfunctional rRNA decay / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network ...proteasome regulatory particle assembly / mitotic cell cycle phase transition / cytosolic proteasome complex / protein-containing complex localization / proteasome-activating activity / proteasome regulatory particle, base subcomplex / cyclin-dependent protein serine/threonine kinase regulator activity / nonfunctional rRNA decay / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Cross-presentation of soluble exogenous antigens (endosomes) / TNFR2 non-canonical NF-kB pathway / proteasomal ubiquitin-independent protein catabolic process / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Regulation of PTEN stability and activity / peptide catabolic process / CDK-mediated phosphorylation and removal of Cdc6 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / KEAP1-NFE2L2 pathway / Neddylation / Orc1 removal from chromatin / MAPK6/MAPK4 signaling / proteasome storage granule / Antigen processing: Ubiquitination & Proteasome degradation / positive regulation of RNA polymerase II transcription preinitiation complex assembly / proteasome core complex, alpha-subunit complex / Ub-specific processing proteases / ERAD pathway / Neutrophil degranulation / proteasome complex / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / positive regulation of protein catabolic process / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / chromatin remodeling / protein domain specific binding / cell division / mRNA binding / ubiquitin protein ligase binding / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / : / 26S proteasome regulatory subunit 7, OB domain / Cyclin, C-terminal domain / Cyclin_C / : ...: / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / : / 26S proteasome regulatory subunit 7, OB domain / Cyclin, C-terminal domain / Cyclin_C / : / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / Cyclin, N-terminal / Cyclin, N-terminal domain / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / Nucleophile aminohydrolases, N-terminal / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
G2/mitotic-specific cyclin-B / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit alpha type-6 ...G2/mitotic-specific cyclin-B / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / 26S proteasome regulatory subunit 4 homolog / 26S proteasome subunit RPT4 / 26S proteasome regulatory subunit 8 homolog
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.27 Å
Authorsde la Pena AH / Goodall EA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM094497 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2-EB020402 United States
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.
History
DepositionAug 15, 2018-
Header (metadata) releaseOct 17, 2018-
Map releaseOct 17, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
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  • Surface view colored by radius
  • Surface level: 0.04
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  • Surface view with fitted model
  • Atomic models: PDB-6ef2
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ef2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9044.png

Downloads & links

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Map

FileDownload / File: emd_9044.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast 26S proteasome bound to ubiquitinated substrate (5T motor state)
Projections & slices

Image control

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

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum0.0 - 0.19236124
Average (Standard dev.)0.0014019879 (±0.007222254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 350.19998 Å
α=β=γ: 90.0 °

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/mean0.0000.1920.001

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Supplemental data

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Mask #1

Fileemd_9044_msk_1.map
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Mask #2

Fileemd_9044_msk_2.map
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Mask #3

Fileemd_9044_msk_3.map
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Additional map: Alpha ring (sharpened)

Fileemd_9044_additional_1.map
AnnotationAlpha ring (sharpened)
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Additional map: Alpha ring (half 1)

Fileemd_9044_additional_2.map
AnnotationAlpha ring (half 1)
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Additional map: Alpha ring (half 2)

Fileemd_9044_additional_3.map
AnnotationAlpha ring (half 2)
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Additional map: Global (half 1)

Fileemd_9044_additional_4.map
AnnotationGlobal (half 1)
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Additional map: Global (sharpened)

Fileemd_9044_additional_5.map
AnnotationGlobal (sharpened)
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Additional map: Global (half 2)

Fileemd_9044_additional_6.map
AnnotationGlobal (half 2)
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Additional map: Motor (half 2)

Fileemd_9044_additional_7.map
AnnotationMotor (half 2)
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Additional map: Motor (half 1)

Fileemd_9044_additional_8.map
AnnotationMotor (half 1)
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Additional map: Motor (sharpened)

Fileemd_9044_additional_9.map
AnnotationMotor (sharpened)
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Sample components

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Entire : Substrate-engaged 26S proteasome in the 5T state

EntireName: Substrate-engaged 26S proteasome in the 5T state
Components
  • Complex: Substrate-engaged 26S proteasome in the 5T state
    • Complex: Proteasome
      • Protein or peptide: Proteasome subunit alpha type-1
      • Protein or peptide: Proteasome subunit alpha type-2
      • Protein or peptide: Proteasome subunit alpha type-3
      • Protein or peptide: Proteasome subunit alpha type-4
      • Protein or peptide: Proteasome subunit alpha type-5
      • Protein or peptide: Proteasome subunit alpha type-6
      • Protein or peptide: Probable proteasome subunit alpha type-7
      • Protein or peptide: 26S proteasome regulatory subunit 7 homolog
      • Protein or peptide: 26S proteasome regulatory subunit 4 homolog
      • Protein or peptide: 26S proteasome regulatory subunit 8 homolog
      • Protein or peptide: 26S proteasome regulatory subunit 6B homolog
      • Protein or peptide: 26S proteasome subunit RPT4
      • Protein or peptide: 26S proteasome regulatory subunit 6A
    • Complex: substrate
      • Protein or peptide: model substrate polypeptide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Substrate-engaged 26S proteasome in the 5T state

SupramoleculeName: Substrate-engaged 26S proteasome in the 5T state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#14
Details: Yeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP

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Supramolecule #2: Proteasome

SupramoleculeName: Proteasome / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#13
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c

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Supramolecule #3: substrate

SupramoleculeName: substrate / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #14
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Proteasome subunit alpha type-1

MacromoleculeName: Proteasome subunit alpha type-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 26.759469 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: AGYDRHITIF SPEGRLYQVE YAFKATNQTN INSLAVRGKD CTVVISQKKV PDKLLDPTTV SYIFCISRTI GMVVNGPIPD ARNAALRAK AEAAEFRYKY GYDMPCDVLA KRMANLSQIY TQRAYMRPLG VILTFVSVDE ELGPSIYKTD PAGYYVGYKA T ATGPKQQE ...String:
AGYDRHITIF SPEGRLYQVE YAFKATNQTN INSLAVRGKD CTVVISQKKV PDKLLDPTTV SYIFCISRTI GMVVNGPIPD ARNAALRAK AEAAEFRYKY GYDMPCDVLA KRMANLSQIY TQRAYMRPLG VILTFVSVDE ELGPSIYKTD PAGYYVGYKA T ATGPKQQE ITTNLENHFK KSKIDHINEE SWEKVVEFAI THMIDALGTE FSKNDLEVGV ATKDKFFTLS AENIEERLVA

UniProtKB: Proteasome subunit alpha type-1

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Macromolecule #2: Proteasome subunit alpha type-2

MacromoleculeName: Proteasome subunit alpha type-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 27.07867 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: MTDRYSFSLT TFSPSGKLGQ IDYALTAVKQ GVTSLGIKAT NGVVIATEKK SSSPLAMSET LSKVSLLTPD IGAVYSGMGP DYRVLVDKS RKVAHTSYKR IYGEYPPTKL LVSEVAKIMQ EATQSGGVRP FGVSLLIAGH DEFNGFSLYQ VDPSGSYFPW K ATAIGKGS ...String:
MTDRYSFSLT TFSPSGKLGQ IDYALTAVKQ GVTSLGIKAT NGVVIATEKK SSSPLAMSET LSKVSLLTPD IGAVYSGMGP DYRVLVDKS RKVAHTSYKR IYGEYPPTKL LVSEVAKIMQ EATQSGGVRP FGVSLLIAGH DEFNGFSLYQ VDPSGSYFPW K ATAIGKGS VAAKTFLEKR WNDELELEDA IHIALLTLKE SVEGEFNGDT IELAIIGDEN PDLLGYTGIP TDKGPRFRKL TS QEINDRL EA

UniProtKB: Proteasome subunit alpha type-2

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Macromolecule #3: Proteasome subunit alpha type-3

MacromoleculeName: Proteasome subunit alpha type-3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 26.805178 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: RRYDSRTTIF SPEGRLYQVE YALESISHAG TAIGIMASDG IVLAAERKVT STLLEQDTST EKLYKLNDKI AVAVAGLTAD AEILINTAR IHAQNYLKTY NEDIPVEILV RRLSDIKQGY TQHGGLRPFG VSFIYAGYDD RYGYQLYTSN PSGNYTGWKA I SVGANTSA ...String:
RRYDSRTTIF SPEGRLYQVE YALESISHAG TAIGIMASDG IVLAAERKVT STLLEQDTST EKLYKLNDKI AVAVAGLTAD AEILINTAR IHAQNYLKTY NEDIPVEILV RRLSDIKQGY TQHGGLRPFG VSFIYAGYDD RYGYQLYTSN PSGNYTGWKA I SVGANTSA AQTLLQMDYK DDMKVDDAIE LALKTLSKTT DSSALTYDRL EFATIRKGAN DGEVYQKIFK PQEIKDILVK TG I

UniProtKB: Proteasome subunit alpha type-3

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Macromolecule #4: Proteasome subunit alpha type-4

MacromoleculeName: Proteasome subunit alpha type-4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 26.862277 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: SGYDRALSIF SPDGHIFQVE YALEAVKRGT CAVGVKGKNC VVLGCERRST LKLQDTRITP SKVSKIDSHV VLSFSGLNAD SRILIEKAR VEAQSHRLTL EDPVTVEYLT RYVAGVQQRY TQSGGVRPFG VSTLIAGFDP RDDEPKLYQT EPSGIYSSWS A QTIGRNSK ...String:
SGYDRALSIF SPDGHIFQVE YALEAVKRGT CAVGVKGKNC VVLGCERRST LKLQDTRITP SKVSKIDSHV VLSFSGLNAD SRILIEKAR VEAQSHRLTL EDPVTVEYLT RYVAGVQQRY TQSGGVRPFG VSTLIAGFDP RDDEPKLYQT EPSGIYSSWS A QTIGRNSK TVREFLEKNY DRKEPPATVE ECVKLTVRSL LEVVQTGAKN IEITVVKPDS DIVALSSEEI NQYVTQIEQE KQ E

UniProtKB: Proteasome subunit alpha type-4

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Macromolecule #5: Proteasome subunit alpha type-5

MacromoleculeName: Proteasome subunit alpha type-5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 27.242596 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: FLTRSEYDRG VSTFSPEGRL FQVEYSLEAI KLGSTAIGIA TKEGVVLGVE KRATSPLLES DSIEKIVEID RHIGCAMSGL TADARSMIE HARTAAVTHN LYYDEDINVE SLTQSVCDLA LRFGEGASGE ERLMSRPFGV ALLIAGHDAD DGYQLFHAEP S GTFYRYNA ...String:
FLTRSEYDRG VSTFSPEGRL FQVEYSLEAI KLGSTAIGIA TKEGVVLGVE KRATSPLLES DSIEKIVEID RHIGCAMSGL TADARSMIE HARTAAVTHN LYYDEDINVE SLTQSVCDLA LRFGEGASGE ERLMSRPFGV ALLIAGHDAD DGYQLFHAEP S GTFYRYNA KAIGSGSEGA QAELLNEWHS SLTLKEAELL VLKILKQVME EKLDENNAQL SCITKQDGFK IYDNEKTAEL IK ELKEKEA

UniProtKB: Proteasome subunit alpha type-5

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Macromolecule #6: Proteasome subunit alpha type-6

MacromoleculeName: Proteasome subunit alpha type-6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 25.355631 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: RNNYDGDTVT FSPTGRLFQV EYALEAIKQG SVTVGLRSNT HAVLVALKRN ADELSSYQKK IIKCDEHMGL SLAGLAPDAR VLSNYLRQQ CNYSSLVFNR KLAVERAGHL LCDKAQKNTQ SYGGRPYGVG LLIIGYDKSG AHLLEFQPSG NVTELYGTAI G ARSQGAKT ...String:
RNNYDGDTVT FSPTGRLFQV EYALEAIKQG SVTVGLRSNT HAVLVALKRN ADELSSYQKK IIKCDEHMGL SLAGLAPDAR VLSNYLRQQ CNYSSLVFNR KLAVERAGHL LCDKAQKNTQ SYGGRPYGVG LLIIGYDKSG AHLLEFQPSG NVTELYGTAI G ARSQGAKT YLERTLDTFI KIDGNPDELI KAGVEAISQS LRDESLTVDN LSIAIVGKDT PFTIYDGEAV AKYI

UniProtKB: Proteasome subunit alpha type-6

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Macromolecule #7: Probable proteasome subunit alpha type-7

MacromoleculeName: Probable proteasome subunit alpha type-7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 27.092719 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: SIGTGYDLSN SVFSPDGRNF QVEYAVKAVE NGTTSIGIKC NDGVVFAVEK LITSKLLVPQ KNVKIQVVDR HIGCVYSGLI PDGRHLVNR GREEAASFKK LYKTPIPIPA FADRLGQYVQ AHTLYNSVRP FGVSTIFGGV DKNGAHLYML EPSGSYWGYK G AATGKGRQ ...String:
SIGTGYDLSN SVFSPDGRNF QVEYAVKAVE NGTTSIGIKC NDGVVFAVEK LITSKLLVPQ KNVKIQVVDR HIGCVYSGLI PDGRHLVNR GREEAASFKK LYKTPIPIPA FADRLGQYVQ AHTLYNSVRP FGVSTIFGGV DKNGAHLYML EPSGSYWGYK G AATGKGRQ SAKAELEKLV DHHPEGLSAR EAVKQAAKII YLAHEDNKEK DFELEISWCS LSETNGLHKF VKGDLLQEAI DF AQKEIN

UniProtKB: Probable proteasome subunit alpha type-7

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Macromolecule #8: 26S proteasome regulatory subunit 7 homolog

MacromoleculeName: 26S proteasome regulatory subunit 7 homolog / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 30.452154 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: SVTMMTVEEK PDVTYSDVGG CKDQIEKLRE VVELPLLSPE RFATLGIDPP KGILLYGPPG TGKTLCARAV ANRTDATFIR VIGSELVQK YVGEGARMVR ELFEMARTKK ACIIFFDEID AVGGARFDDG AGGDNEVQRT MLELITQLDG FDPRGNIKVM F ATNRPNTL ...String:
SVTMMTVEEK PDVTYSDVGG CKDQIEKLRE VVELPLLSPE RFATLGIDPP KGILLYGPPG TGKTLCARAV ANRTDATFIR VIGSELVQK YVGEGARMVR ELFEMARTKK ACIIFFDEID AVGGARFDDG AGGDNEVQRT MLELITQLDG FDPRGNIKVM F ATNRPNTL DPALLRPGRI DRKVEFSLPD LEGRANIFRI HSKSMSVERG IRWELISRLC PNSTGAELRS VCTEAGMFAI RA RRKVATE KDFLKAVDKV ISGYKKFSST SRYMQY

UniProtKB: 26S proteasome regulatory subunit 7 homolog

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Macromolecule #9: 26S proteasome regulatory subunit 4 homolog

MacromoleculeName: 26S proteasome regulatory subunit 4 homolog / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 28.889051 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: TESYSDIGGL ESQIQEIKES VELPLTHPEL YEEMGIKPPK GVILYGAPGT GKTLLAKAVA NQTSATFLRI VGSELIQKYL GDGPRLCRQ IFKVAGENAP SIVFIDEIDA IGTKRYDSNS GGEREIQRTM LELLNQLDGF DDRGDVKVIM ATNKIETLDP A LIRPGRID ...String:
TESYSDIGGL ESQIQEIKES VELPLTHPEL YEEMGIKPPK GVILYGAPGT GKTLLAKAVA NQTSATFLRI VGSELIQKYL GDGPRLCRQ IFKVAGENAP SIVFIDEIDA IGTKRYDSNS GGEREIQRTM LELLNQLDGF DDRGDVKVIM ATNKIETLDP A LIRPGRID RKILFENPDL STKKKILGIH TSKMNLSEDV NLETLVTTKD DLSGADIQAM CTEAGLLALR ERRMQVTAED FK QAKERVM KNKVEENLEG LYL

UniProtKB: 26S proteasome regulatory subunit 4 homolog

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Macromolecule #10: 26S proteasome regulatory subunit 8 homolog

MacromoleculeName: 26S proteasome regulatory subunit 8 homolog / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 29.010736 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: DSTYDMVGGL TKQIKEIKEV IELPVKHPEL FESLGIAQPK GVILYGPPGT GKTLLARAVA HHTDCKFIRV SGAELVQKYI GEGSRMVRE LFVMAREHAP SIIFMDEIDS IGSTRVEGSG GGDSEVQRTM LELLNQLDGF ETSKNIKIIM ATNRLDILDP A LLRPGRID ...String:
DSTYDMVGGL TKQIKEIKEV IELPVKHPEL FESLGIAQPK GVILYGPPGT GKTLLARAVA HHTDCKFIRV SGAELVQKYI GEGSRMVRE LFVMAREHAP SIIFMDEIDS IGSTRVEGSG GGDSEVQRTM LELLNQLDGF ETSKNIKIIM ATNRLDILDP A LLRPGRID RKIEFPPPSV AARAEILRIH SRKMNLTRGI NLRKVAEKMN GCSGADVKGV CTEAGMYALR ERRIHVTQED FE LAVGKVM NKNQETAISV AKLFK

UniProtKB: 26S proteasome regulatory subunit 8 homolog

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Macromolecule #11: 26S proteasome regulatory subunit 6B homolog

MacromoleculeName: 26S proteasome regulatory subunit 6B homolog / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 29.000164 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: TYADVGGLDM QKQEIREAVE LPLVQADLYE QIGIDPPRGV LLYGPPGTGK TMLVKAVANS TKAAFIRVNG SEFVHKYLGE GPRMVRDVF RLARENAPSI IFIDEVDSIA TKRFDAQTGS DREVQRILIE LLTQMDGFDQ STNVKVIMAT NRADTLDPAL L RPGRLDRK ...String:
TYADVGGLDM QKQEIREAVE LPLVQADLYE QIGIDPPRGV LLYGPPGTGK TMLVKAVANS TKAAFIRVNG SEFVHKYLGE GPRMVRDVF RLARENAPSI IFIDEVDSIA TKRFDAQTGS DREVQRILIE LLTQMDGFDQ STNVKVIMAT NRADTLDPAL L RPGRLDRK IEFPSLRDRR ERRLIFGTIA SKMSLAPEAD LDSLIIRNDS LSGAVIAAIM QEAGLRAVRK NRYVILQSDL EE AYATQVK TDNTVDKFDF YK

UniProtKB: 26S proteasome regulatory subunit 6B homolog

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Macromolecule #12: 26S proteasome subunit RPT4

MacromoleculeName: 26S proteasome subunit RPT4 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 29.353756 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: LVYNMTSFEQ GEITFDGIGG LTEQIRELRE VIELPLKNPE IFQRVGIKPP KGVLLYGPPG TGKTLLAKAV AATIGANFIF SPASGIVDK YIGESARIIR EMFAYAKEHE PCIIFMDEVD AIGGRRFSEG TSADREIQRT LMELLTQMDG FDNLGQTKII M ATNRPDTL ...String:
LVYNMTSFEQ GEITFDGIGG LTEQIRELRE VIELPLKNPE IFQRVGIKPP KGVLLYGPPG TGKTLLAKAV AATIGANFIF SPASGIVDK YIGESARIIR EMFAYAKEHE PCIIFMDEVD AIGGRRFSEG TSADREIQRT LMELLTQMDG FDNLGQTKII M ATNRPDTL DPALLRPGRL DRKVEIPLPN EAGRLEIFKI HTAKVKKTGE FDFEAAVKMS DGFNGADIRN CATEAGFFAI RD DRDHINP DDLMKAVRKV AEVKKLE

UniProtKB: 26S proteasome subunit RPT4

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Macromolecule #13: 26S proteasome regulatory subunit 6A

MacromoleculeName: 26S proteasome regulatory subunit 6A / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 29.845061 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae S288c (yeast)
SequenceString: SRVKAMEVDE KPTETYSDVG GLDKQIEELV EAIVLPMKRA DKFKDMGIRA PKGALMYGPP GTGKTLLARA CAAQTNATFL KLAAPQLVQ MYIGEGAKLV RDAFALAKEK APTIIFIDEL DAIGTKRFDS EKSGDREVQR TMLELLNQLD GFSSDDRVKV L AATNRVDV ...String:
SRVKAMEVDE KPTETYSDVG GLDKQIEELV EAIVLPMKRA DKFKDMGIRA PKGALMYGPP GTGKTLLARA CAAQTNATFL KLAAPQLVQ MYIGEGAKLV RDAFALAKEK APTIIFIDEL DAIGTKRFDS EKSGDREVQR TMLELLNQLD GFSSDDRVKV L AATNRVDV LDPALLRSGR LDRKIEFPLP SEDSRAQILQ IHSRKMTTDD DINWQELARS TDEFNGAQLK AVTVEAGMIA LR NGQSSVK HEDFVEGISE VQARKSKSVS FYA

UniProtKB: 26S proteasome regulatory subunit 6A

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Macromolecule #14: model substrate polypeptide

MacromoleculeName: model substrate polypeptide / type: protein_or_peptide / ID: 14 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.572701 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
NNENVSARLG GASIAV

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Macromolecule #15: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 15 / Number of copies: 4 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #16: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 16 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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

Concentration25 mg/mL
BufferpH: 7.6
Component:
ConcentrationName
20.0 mMHEPES
25.0 mMNaCl
25.0 mMKCl
10.0 mMMgCl2
1.0 mMTCEP
5.0 mMATP
0.05 % (w/v)Nonidet P-40
6.0 mMortho-phenanthroline
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: specimens were manually blotted with Whatman #1 filter paper.
Details26S proteasomes were diluted to a concentration of 20 micromolar in a buffer with an ATP regeneration system, and 6 mM ortho-phenanthroline. This solution was mixed with an equal volume of 50 micromolar ubiquitinated model substrate

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Detailsimages were acquired in nanoprobe mode
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-25 / Number grids imaged: 1 / Number real images: 11656 / Average exposure time: 6.25 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: -3.0 µm / Calibrated defocus min: -1.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 29000
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

DetailsCamera was operated in counting mode
Particle selectionNumber selected: 579361
Details: Particles were selected using the Relion template-based particle picker
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5mp9

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 47349
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5mpc


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6ef2:
Yeast 26S proteasome bound to ubiquitinated substrate (5T motor state)

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