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

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Basic information

Entry
Database: EMDB / ID: EMD-9042
TitleYeast 26S proteasome bound to ubiquitinated substrate (1D* motor state)
Map dataYeast 26S proteasome bound to ubiquitinated substrate (1D* motor state)
Sample
  • Complex: Substrate-engaged 26S proteasome in the 1D* 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


mitotic cell cycle phase transition / proteasome regulatory particle assembly / protein-containing complex localization / proteasome-activating activity / proteasome regulatory particle, base subcomplex / cyclin-dependent protein serine/threonine kinase regulator activity / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Proteasome assembly / Cross-presentation of soluble exogenous antigens (endosomes) ...mitotic cell cycle phase transition / proteasome regulatory particle assembly / protein-containing complex localization / proteasome-activating activity / proteasome regulatory particle, base subcomplex / cyclin-dependent protein serine/threonine kinase regulator activity / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Proteasome assembly / 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 / nonfunctional rRNA decay / CDK-mediated phosphorylation and removal of Cdc6 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / peptide catabolic process / 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 / Ub-specific processing proteases / proteasome core complex, alpha-subunit complex / 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 / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Proteasome subunit alpha6 / Proteasome subunit alpha5 / 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 / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / 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: 4.43 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: 0.03
  • 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 modelsPDB-6ef0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9042.png

Downloads & links

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Map

FileDownload / File: emd_9042.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast 26S proteasome bound to ubiquitinated substrate (1D* motor state)
Projections & slices

Image control

Size
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Others
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: 0.03 / Movie #1: 0.03
Minimum - Maximum0.0 - 0.19451101
Average (Standard dev.)0.0012396917 (±0.006442988)
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/mean0.0000.1950.001

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Supplemental data

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Mask #1

Fileemd_9042_msk_1.map
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Mask #2

Fileemd_9042_msk_2.map
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Mask #3

Fileemd_9042_msk_3.map
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Additional map: Motor (half 1)

Fileemd_9042_additional_1.map
AnnotationMotor (half 1)
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Additional map: Motor (sharpened)

Fileemd_9042_additional_2.map
AnnotationMotor (sharpened)
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Additional map: Global (half 2)

Fileemd_9042_additional_3.map
AnnotationGlobal (half 2)
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Additional map: Global (half 1)

Fileemd_9042_additional_4.map
AnnotationGlobal (half 1)
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Additional map: Alpha ring (half 1)

Fileemd_9042_additional_5.map
AnnotationAlpha ring (half 1)
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Additional map: Alpha ring (sharpened)

Fileemd_9042_additional_6.map
AnnotationAlpha ring (sharpened)
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Additional map: Global (sharpened)

Fileemd_9042_additional_7.map
AnnotationGlobal (sharpened)
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Additional map: Alpha ring (half 2)

Fileemd_9042_additional_8.map
AnnotationAlpha ring (half 2)
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Additional map: Motor (half 2)

Fileemd_9042_additional_9.map
AnnotationMotor (half 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
Components
  • Complex: Substrate-engaged 26S proteasome in the 1D* 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'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Substrate-engaged 26S proteasome in the 1D* state

SupramoleculeName: Substrate-engaged 26S proteasome in the 1D* 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: 26.801549 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: GYDRHITIFS PEGRLYQVEY AFKATNQTNI NSLAVRGKDC TVVISQKKVP DKLLDPTTVS YIFCISRTIG MVVNGPIPDA RNAALRAKA EAAEFRYKYG YDMPCDVLAK RMANLSQIYT QRAYMRPLGV ILTFVSVDEE LGPSIYKTDP AGYYVGYKAT A TGPKQQEI ...String:
GYDRHITIFS PEGRLYQVEY AFKATNQTNI NSLAVRGKDC TVVISQKKVP DKLLDPTTVS YIFCISRTIG MVVNGPIPDA RNAALRAKA EAAEFRYKYG YDMPCDVLAK RMANLSQIYT QRAYMRPLGV ILTFVSVDEE LGPSIYKTDP AGYYVGYKAT A TGPKQQEI TTNLENHFKK SKIDHINEES WEKVVEFAIT HMIDALGTEF SKNDLEVGVA TKDKFFTLSA ENIEERLVAI

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: 27.191828 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (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 EAL

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: 27.080506 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MGSRRYDSRT TIFSPEGRLY QVEYALESIS HAGTAIGIMA SDGIVLAAER KVTSTLLEQD TSTEKLYKLN DKIAVAVAGL TADAEILIN TARIHAQNYL KTYNEDIPVE ILVRRLSDIK QGYTQHGGLR PFGVSFIYAG YDDRYGYQLY TSNPSGNYTG W KAISVGAN ...String:
MGSRRYDSRT TIFSPEGRLY QVEYALESIS HAGTAIGIMA SDGIVLAAER KVTSTLLEQD TSTEKLYKLN DKIAVAVAGL TADAEILIN TARIHAQNYL KTYNEDIPVE ILVRRLSDIK QGYTQHGGLR PFGVSFIYAG YDDRYGYQLY TSNPSGNYTG W KAISVGAN TSAAQTLLQM DYKDDMKVDD AIELALKTLS KTTDSSALTY DRLEFATIRK GANDGEVYQK IFKPQEIKDI LV KTGI

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: 26.993475 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MSGYDRALSI FSPDGHIFQV EYALEAVKRG TCAVGVKGKN CVVLGCERRS TLKLQDTRIT PSKVSKIDSH VVLSFSGLNA DSRILIEKA RVEAQSHRLT LEDPVTVEYL TRYVAGVQQR YTQSGGVRPF GVSTLIAGFD PRDDEPKLYQ TEPSGIYSSW S AQTIGRNS ...String:
MSGYDRALSI FSPDGHIFQV EYALEAVKRG TCAVGVKGKN CVVLGCERRS TLKLQDTRIT PSKVSKIDSH VVLSFSGLNA DSRILIEKA RVEAQSHRLT LEDPVTVEYL TRYVAGVQQR YTQSGGVRPF GVSTLIAGFD PRDDEPKLYQ TEPSGIYSSW S AQTIGRNS KTVREFLEKN YDRKEPPATV EECVKLTVRS LLEVVQTGAK NIEITVVKPD SDIVALSSEE INQYVTQIEQ EK QE

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: 27.444869 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MFLTRSEYDR GVSTFSPEGR LFQVEYSLEA IKLGSTAIGI ATKEGVVLGV EKRATSPLLE SDSIEKIVEI DRHIGCAMSG LTADARSMI EHARTAAVTH NLYYDEDINV ESLTQSVCDL ALRFGEGASG EERLMSRPFG VALLIAGHDA DDGYQLFHAE P SGTFYRYN ...String:
MFLTRSEYDR GVSTFSPEGR LFQVEYSLEA IKLGSTAIGI ATKEGVVLGV EKRATSPLLE SDSIEKIVEI DRHIGCAMSG LTADARSMI EHARTAAVTH NLYYDEDINV ESLTQSVCDL ALRFGEGASG EERLMSRPFG VALLIAGHDA DDGYQLFHAE P SGTFYRYN AKAIGSGSEG AQAELLNEWH SSLTLKEAEL LVLKILKQVM EEKLDENNAQ LSCITKQDGF KIYDNEKTAE LI KELKEKE AA

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: 25.634 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MFRNNYDGDT VTFSPTGRLF QVEYALEAIK QGSVTVGLRS NTHAVLVALK RNADELSSYQ KKIIKCDEHM GLSLAGLAPD ARVLSNYLR QQCNYSSLVF NRKLAVERAG HLLCDKAQKN TQSYGGRPYG VGLLIIGYDK SGAHLLEFQP SGNVTELYGT A IGARSQGA ...String:
MFRNNYDGDT VTFSPTGRLF QVEYALEAIK QGSVTVGLRS NTHAVLVALK RNADELSSYQ KKIIKCDEHM GLSLAGLAPD ARVLSNYLR QQCNYSSLVF NRKLAVERAG HLLCDKAQKN TQSYGGRPYG VGLLIIGYDK SGAHLLEFQP SGNVTELYGT A IGARSQGA KTYLERTLDT FIKIDGNPDE LIKAGVEAIS QSLRDESLTV DNLSIAIVGK DTPFTIYDGE AVAKYI

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 W303 (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: 28.597023 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: EEKPDVTYSD VGGCKDQIEK LREVVELPLL SPERFATLGI DPPKGILLYG PPGTGKTLCA RAVANRTDAT FIRVIGSELV QKYVGEGAR MVRELFEMAR TKKACIIFFD EIDAVGGARF DDGAGGDNEV QRTMLELITQ LDGFDPRGNI KVMFATNRPN T LDPALLRP ...String:
EEKPDVTYSD VGGCKDQIEK LREVVELPLL SPERFATLGI DPPKGILLYG PPGTGKTLCA RAVANRTDAT FIRVIGSELV QKYVGEGAR MVRELFEMAR TKKACIIFFD EIDAVGGARF DDGAGGDNEV QRTMLELITQ LDGFDPRGNI KVMFATNRPN T LDPALLRP GRIDRKVEFS LPDLEGRANI FRIHSKSMSV ERGIRWELIS RLCPNSTGAE LRSVCTEAGM FAIRARRKVA TE KDFLKAV DKVISGYKKF

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: 30.109572 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: PMVSVMKMDK SPTESYSDIG GLESQIQEIK ESVELPLTHP ELYEEMGIKP PKGVILYGAP GTGKTLLAKA VANQTSATFL RIVGSELIQ KYLGDGPRLC RQIFKVAGEN APSIVFIDEI DAIGTKRYDS NSGGEREIQR TMLELLNQLD GFDDRGDVKV I MATNKIET ...String:
PMVSVMKMDK SPTESYSDIG GLESQIQEIK ESVELPLTHP ELYEEMGIKP PKGVILYGAP GTGKTLLAKA VANQTSATFL RIVGSELIQ KYLGDGPRLC RQIFKVAGEN APSIVFIDEI DAIGTKRYDS NSGGEREIQR TMLELLNQLD GFDDRGDVKV I MATNKIET LDPALIRPGR IDRKILFENP DLSTKKKILG IHTSKMNLSE DVNLETLVTT KDDLSGADIQ AMCTEAGLLA LR ERRMQVT AEDFKQAKER VMKNKVEENL EGLY

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: 30.522594 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: ADPLVSLMMV EKVPDSTYDM VGGLTKQIKE IKEVIELPVK HPELFESLGI AQPKGVILYG PPGTGKTLLA RAVAHHTDCK FIRVSGAEL VQKYIGEGSR MVRELFVMAR EHAPSIIFMD EIDSIGSTRV EGSGGGDSEV QRTMLELLNQ LDGFETSKNI K IIMATNRL ...String:
ADPLVSLMMV EKVPDSTYDM VGGLTKQIKE IKEVIELPVK HPELFESLGI AQPKGVILYG PPGTGKTLLA RAVAHHTDCK FIRVSGAEL VQKYIGEGSR MVRELFVMAR EHAPSIIFMD EIDSIGSTRV EGSGGGDSEV QRTMLELLNQ LDGFETSKNI K IIMATNRL DILDPALLRP GRIDRKIEFP PPSVAARAEI LRIHSRKMNL TRGINLRKVA EKMNGCSGAD VKGVCTEAGM YA LRERRIH VTQEDFELAV GKVMNKNQET AISVAKLFK

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: 30.387693 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: SISVMGENEK PDVTYADVGG LDMQKQEIRE AVELPLVQAD LYEQIGIDPP RGVLLYGPPG TGKTMLVKAV ANSTKAAFIR VNGSEFVHK YLGEGPRMVR DVFRLARENA PSIIFIDEVD SIATKRFDAQ TGSDREVQRI LIELLTQMDG FDQSTNVKVI M ATNRADTL ...String:
SISVMGENEK PDVTYADVGG LDMQKQEIRE AVELPLVQAD LYEQIGIDPP RGVLLYGPPG TGKTMLVKAV ANSTKAAFIR VNGSEFVHK YLGEGPRMVR DVFRLARENA PSIIFIDEVD SIATKRFDAQ TGSDREVQRI LIELLTQMDG FDQSTNVKVI M ATNRADTL DPALLRPGRL DRKIEFPSLR DRRERRLIFG TIASKMSLAP EADLDSLIIR NDSLSGAVIA AIMQEAGLRA VR KNRYVIL QSDLEEAYAT QVKTDNTVDK FDFYK

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: 30.386865 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: DPLVYNMTSF EQGEITFDGI GGLTEQIREL REVIELPLKN PEIFQRVGIK PPKGVLLYGP PGTGKTLLAK AVAATIGANF IFSPASGIV DKYIGESARI IREMFAYAKE HEPCIIFMDE VDAIGGRRFS EGTSADREIQ RTLMELLTQM DGFDNLGQTK I IMATNRPD ...String:
DPLVYNMTSF EQGEITFDGI GGLTEQIREL REVIELPLKN PEIFQRVGIK PPKGVLLYGP PGTGKTLLAK AVAATIGANF IFSPASGIV DKYIGESARI IREMFAYAKE HEPCIIFMDE VDAIGGRRFS EGTSADREIQ RTLMELLTQM DGFDNLGQTK I IMATNRPD TLDPALLRPG RLDRKVEIPL PNEAGRLEIF KIHTAKVKKT GEFDFEAAVK MSDGFNGADI RNCATEAGFF AI RDDRDHI NPDDLMKAVR KVAEVKKLEG TIEYQK

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: 28.497504 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: PTETYSDVGG LDKQIEELVE AIVLPMKRAD KFKDMGIRAP KGALMYGPPG TGKTLLARAC AAQTNATFLK LAAPQLVQMY IGEGAKLVR DAFALAKEKA PTIIFIDELD AIGTKRFDSE KSGDREVQRT MLELLNQLDG FSSDDRVKVL AATNRVDVLD P ALLRSGRL ...String:
PTETYSDVGG LDKQIEELVE AIVLPMKRAD KFKDMGIRAP KGALMYGPPG TGKTLLARAC AAQTNATFLK LAAPQLVQMY IGEGAKLVR DAFALAKEKA PTIIFIDELD AIGTKRFDSE KSGDREVQRT MLELLNQLDG FSSDDRVKVL AATNRVDVLD P ALLRSGRL DRKIEFPLPS EDSRAQILQI HSRKMTTDDD INWQELARST DEFNGAQLKA VTVEAGMIAL RNGQSSVKHE DF VEGISEV QARKSKSVSF Y

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: 1.130277 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
SARLGGASIA VQ

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Macromolecule #15: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 15 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #16: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 16 / Number of copies: 3 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, 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
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
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) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 34701
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-6ef0:
Yeast 26S proteasome bound to ubiquitinated substrate (1D* motor state)

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