EMDB-9045: Yeast 26S proteasome bound to ubiquitinated substrate (4D motor state)

Basic information

• Entry: Database: EMDB / ID: EMD-9045
• Title: Yeast 26S proteasome bound to ubiquitinated substrate (4D motor state)
• Map data: Yeast 26S proteasome bound to ubiquitinated substrate (4D motor state)

Sample

• Complex: Substrate-engaged 26S proteasome in the 4D state
  • Complex: Proteasome
    • Protein or peptide: x 23 types
  • Complex: substrate
    • Protein or peptide: x 1 types
• Ligand: x 2 types

• Keywords: 26S Proteasome / ATPase / AAA+ / Protease / Motor protein / Ubiquitin

Function / homology

Function:

mitotic cell cycle phase transition / Metalloprotease DUBs / proteasome regulatory particle assembly / proteasome storage granule assembly / peroxisome fission / protein-containing complex localization / proteasome-activating activity / mitochondrial fission / proteasome regulatory particle, lid subcomplex / proteasome regulatory particle, base subcomplex / metal-dependent deubiquitinase activity / 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 / proteasome binding / Orc1 removal from chromatin / MAPK6/MAPK4 signaling / proteasome storage granule / Antigen processing: Ubiquitination & Proteasome degradation / ribosomal large subunit export from nucleus / positive regulation of RNA polymerase II transcription preinitiation complex assembly / protein deubiquitination / endopeptidase activator activity / Ub-specific processing proteases / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome assembly / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / ERAD pathway / Neutrophil degranulation / proteasome complex / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / modification-dependent protein catabolic process / protein tag activity / positive regulation of protein catabolic process / metallopeptidase activity / peroxisome / ribosome biogenesis / ribosomal large subunit assembly / ubiquitin-dependent protein catabolic process / endopeptidase activity / cytosolic large ribosomal subunit / proteasome-mediated ubiquitin-dependent protein catabolic process / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / cytoplasmic translation / protein ubiquitination / structural constituent of ribosome / chromatin remodeling / protein domain specific binding / cell division / mRNA binding / ubiquitin protein ligase binding / endoplasmic reticulum membrane / ATP hydrolysis activity / mitochondrion / ATP binding / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm

Domain/homology:

: / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / : / 26S proteasome regulatory subunit RPN11 C-terminal domain / : / 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 / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / 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 B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / AAA ATPase, AAA+ lid domain / AAA+ lid domain / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / MPN domain / MPN domain profile. / Nucleophile aminohydrolases, N-terminal / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase

Component:

G2/mitotic-specific cyclin-B / Ubiquitin-ribosomal protein eL40A fusion protein / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-4 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit beta type-6 / Proteasome subunit beta type-2 / Proteasome subunit beta type-3 / Proteasome subunit beta type-5 / Proteasome subunit beta type-7 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit beta type-1 / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / 26S proteasome regulatory subunit 4 homolog / Ubiquitin carboxyl-terminal hydrolase RPN11 / 26S proteasome subunit RPT4 / 26S proteasome regulatory subunit 8 homolog

• Biological species: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Homo sapiens (human) / Saccharomyces cerevisiae S288c (yeast)
• Method: single particle reconstruction / cryo EM / Resolution: 4.17 Å
• Authors: de la Pena AH / Goodall EA

Funding support

United States, 2 items

Organization	Grant number	Country
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

• Citation: Journal: 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 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

Deposition	Aug 15, 2018	-
Header (metadata) release	Oct 17, 2018	-
Map release	Oct 17, 2018	-
Update	Oct 30, 2024	-
Current status	Oct 30, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_9045.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Yeast 26S proteasome bound to ubiquitinated substrate (4D motor state)
• Voxel size: X=Y=Z: 1.03 Å

Density

Contour Level	By AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum	0.0 - 0.22217602
Average (Standard dev.)	0.001485774 (±0.008148518)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 340 340 340 Spacing 340 340 340
Cell	A=B=C: 350.19998 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.03	1.03	1.03
M x/y/z	340	340	340
origin x/y/z	0.000	0.000	0.000
length x/y/z	350.200	350.200	350.200
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	450	450	450
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	340	340	340
D min/max/mean	0.000	0.222	0.001

Supplemental data

Mask #1

• File: emd_9045_msk_1.map

Mask #2

• File: emd_9045_msk_2.map

Mask #3

• File: emd_9045_msk_3.map

Mask #4

• File: emd_9045_msk_4.map

Mask #5

• File: emd_9045_msk_5.map

Mask #6

• File: emd_9045_msk_6.map

Additional map: Global (sharpened)

• File: emd_9045_additional_1.map
• Annotation: Global (sharpened)

Additional map: Alpha ring (sharpened)

• File: emd_9045_additional_10.map
• Annotation: Alpha ring (sharpened)

Additional map: Alpha ring (half 1)

• File: emd_9045_additional_11.map
• Annotation: Alpha ring (half 1)

Additional map: Alpha ring (half 2)

• File: emd_9045_additional_12.map
• Annotation: Alpha ring (half 2)

Additional map: Beta ring (sharpened)

• File: emd_9045_additional_13.map
• Annotation: Beta ring (sharpened)

Additional map: Beta ring (half 1)

• File: emd_9045_additional_14.map
• Annotation: Beta ring (half 1)

Additional map: Beta ring (half 2)

• File: emd_9045_additional_15.map
• Annotation: Beta ring (half 2)

Additional map: Motor (sharpened)

• File: emd_9045_additional_16.map
• Annotation: Motor (sharpened)

Additional map: Motor (half 1)

• File: emd_9045_additional_17.map
• Annotation: Motor (half 1)

Additional map: Motor (half 2)

• File: emd_9045_additional_18.map
• Annotation: Motor (half 2)

Additional map: Global (half 1)

• File: emd_9045_additional_2.map
• Annotation: Global (half 1)

Additional map: Global (half 2)

• File: emd_9045_additional_3.map
• Annotation: Global (half 2)

Additional map: Rpn11 (sharpened)

• File: emd_9045_additional_4.map
• Annotation: Rpn11 (sharpened)

Additional map: Rpn11 (half 1)

• File: emd_9045_additional_5.map
• Annotation: Rpn11 (half 1)

Additional map: Rpn11 (half 2)

• File: emd_9045_additional_6.map
• Annotation: Rpn11 (half 2)

Additional map: N-ring (sharpened)

• File: emd_9045_additional_7.map
• Annotation: N-ring (sharpened)

Additional map: N-ring (half 1)

• File: emd_9045_additional_8.map
• Annotation: N-ring (half 1)

Additional map: N-ring (half 2)

• File: emd_9045_additional_9.map
• Annotation: N-ring (half 2)

Sample components

Entire : Substrate-engaged 26S proteasome in the 4D state

• Entire: Name: Substrate-engaged 26S proteasome in the 4D state

Components

• Complex: Substrate-engaged 26S proteasome in the 4D state
  • Complex: Proteasome
    • Protein or peptide: Proteasome subunit beta type-1
    • Protein or peptide: Proteasome subunit beta type-2
    • Protein or peptide: Proteasome subunit beta type-3
    • Protein or peptide: Proteasome subunit beta type-4
    • Protein or peptide: Proteasome subunit beta type-5
    • Protein or peptide: Proteasome subunit beta type-6
    • Protein or peptide: Proteasome subunit beta type-7
    • 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
    • Protein or peptide: Proteasome subunit beta type-7
    • Protein or peptide: Ubiquitin carboxyl-terminal hydrolase RPN11
    • Protein or peptide: Model substrate polypeptide
  • Complex: substrate
    • Protein or peptide: Ubiquitin-60S ribosomal protein L40
• Ligand: ADENOSINE-5'-TRIPHOSPHATE
• Ligand: ADENOSINE-5'-DIPHOSPHATE

Supramolecule #1: Substrate-engaged 26S proteasome in the 4D state

• Supramolecule: Name: Substrate-engaged 26S proteasome in the 4D state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#24; Details: Yeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP

Supramolecule #2: Proteasome

• Supramolecule: Name: Proteasome / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#23
• Source (natural): Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast); Strain: ATCC 204508 / S288c

Supramolecule #3: substrate

• Supramolecule: Name: substrate / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #24
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: Proteasome subunit beta type-1

• Macromolecule: Name: Proteasome subunit beta type-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 23.573604 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MNGIQVDINR LKKGEVSLGT SIMAVTFKDG VILGADSRTT TGAYIANRVT DKLTRVHDKI WCCRSGSAAD TQAIADIVQY HLELYTSQY GTPSTETAAS VFKELCYENK DNLTAGIIVA GYDDKNKGEV YTIPLGGSVH KLPYAIAGSG STFIYGYCDK N FRENMSKE ETVDFIKHSL SQAIKWDGSS GGVIRMVVLT AAGVERLIFY PDEYEQL

UniProtKB: Proteasome subunit beta type-1

Macromolecule #2: Proteasome subunit beta type-2

• Macromolecule: Name: Proteasome subunit beta type-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 28.299889 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MAGLSFDNYQ RNNFLAENSH TQPKATSTGT TIVGVKFNNG VVIAADTRST QGPIVADKNC AKLHRISPKI WCAGAGTAAD TEAVTQLIG SNIELHSLYT SREPRVVSAL QMLKQHLFKY QGHIGAYLIV AGVDPTGSHL FSIHAHGSTD VGYYLSLGSG S LAAMAVLE SHWKQDLTKE EAIKLASDAI QAGIWNDLGS GSNVDVCVME IGKDAEYLRN YLTPNVREEK QKSYKFPRGT TA VLKESIV NICDIQEEQV DITA

UniProtKB: Proteasome subunit beta type-2

Macromolecule #3: Proteasome subunit beta type-3

• Macromolecule: Name: Proteasome subunit beta type-3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 22.627842 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MSDPSSINGG IVVAMTGKDC VAIACDLRLG SQSLGVSNKF EKIFHYGHVF LGITGLATDV TTLNEMFRYK TNLYKLKEER AIEPETFTQ LVSSSLYERR FGPYFVGPVV AGINSKSGKP FIAGFDLIGC IDEAKDFIVS GTASDQLFGM CESLYEPNLE P EDLFETIS QALLNAADRD ALSGWGAVVY IIKKDEVVKR YLKMRQD

UniProtKB: Proteasome subunit beta type-3

Macromolecule #4: Proteasome subunit beta type-4

• Macromolecule: Name: Proteasome subunit beta type-4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 22.545676 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MDIILGIRVQ DSVILASSKA VTRGISVLKD SDDKTRQLSP HTLMSFAGEA GDTVQFAEYI QANIQLYSIR EDYELSPQAV SSFVRQELA KSIRSRRPYQ VNVLIGGYDK KKNKPELYQI DYLGTKVELP YGAHGYSGFY TFSLLDHHYR PDMTTEEGLD L LKLCVQEL EKRMPMDFKG VIVKIVDKDG IRQVDDFQAQ

UniProtKB: Proteasome subunit beta type-4

Macromolecule #5: Proteasome subunit beta type-5

• Macromolecule: Name: Proteasome subunit beta type-5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 31.670539 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MQAIADSFSV PNRLVKELQY DNEQNLESDF VTGASQFQRL APSLTVPPIA SPQQFLRAHT DDSRNPDCKI KIAHGTTTLA FRFQGGIIV AVDSRATAGN WVASQTVKKV IEINPFLLGT MAGGAADCQF WETWLGSQCR LHELREKERI SVAAASKILS N LVYQYKGA GLSMGTMICG YTRKEGPTIY YVDSDGTRLK GDIFCVGSGQ TFAYGVLDSN YKWDLSVEDA LYLGKRSILA AA HRDAYSG GSVNLYHVTE DGWIYHGNHD VGELFWKVKE EEGSFNNVIG

UniProtKB: Proteasome subunit beta type-5

Macromolecule #6: Proteasome subunit beta type-6

• Macromolecule: Name: Proteasome subunit beta type-6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 26.905076 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MATIASEYSS EASNTPIEHQ FNPYGDNGGT ILGIAGEDFA VLAGDTRNIT DYSINSRYEP KVFDCGDNIV MSANGFAADG DALVKRFKN SVKWYHFDHN DKKLSINSAA RNIQHLLYGK RFFPYYVHTI IAGLDEDGKG AVYSFDPVGS YEREQCRAGG A AASLIMPF LDNQVNFKNQ YEPGTNGKVK KPLKYLSVEE VIKLVRDSFT SATERHIQVG DGLEILIVTK DGVRKEFYEL KR D

UniProtKB: Proteasome subunit beta type-6

Macromolecule #7: Proteasome subunit beta type-7

• Macromolecule: Name: Proteasome subunit beta type-7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 29.471289 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MNHDPFSWGR PADSTYGAYN TQIANAGASP MVNTQQPIVT GTSVISMKYD NGVIIAADNL GSYGSLLRFN GVERLIPVGD NTVVGISGD ISDMQHIERL LKDLVTENAY DNPLADAEEA LEPSYIFEYL ATVMYQRRSK MNPLWNAIIV AGVQSNGDQF L RYVNLLGV TYSSPTLATG FGAHMANPLL RKVVDRESDI PKTTVQVAEE AIVNAMRVLY YRDARSSRNF SLAIIDKNTG LT FKKNLQV ENMKWDFAKD IKGYGTQKI

UniProtKB: Proteasome subunit beta type-7

Macromolecule #8: Proteasome subunit alpha type-1

• Macromolecule: Name: Proteasome subunit alpha type-1 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 28.03383 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MSGAAAASAA GYDRHITIFS PEGRLYQVEY AFKATNQTNI NSLAVRGKDC TVVISQKKVP DKLLDPTTVS YIFCISRTIG MVVNGPIPD ARNAALRAKA EAAEFRYKYG YDMPCDVLAK RMANLSQIYT QRAYMRPLGV ILTFVSVDEE LGPSIYKTDP A GYYVGYKA TATGPKQQEI TTNLENHFKK SKIDHINEES WEKVVEFAIT HMIDALGTEF SKNDLEVGVA TKDKFFTLSA EN IEERLVA IAEQD

UniProtKB: Proteasome subunit alpha type-1

Macromolecule #9: Proteasome subunit alpha type-2

• Macromolecule: Name: Proteasome subunit alpha type-2 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 27.191828 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

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

Macromolecule #10: Proteasome subunit alpha type-3

• Macromolecule: Name: Proteasome subunit alpha type-3 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 28.74823 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

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 KTGITKK DEDEEADEDM K

UniProtKB: Proteasome subunit alpha type-3

Macromolecule #11: Proteasome subunit alpha type-4

• Macromolecule: Name: Proteasome subunit alpha type-4 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 28.478111 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

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 QEQQEQD KKKKSNH

UniProtKB: Proteasome subunit alpha type-4

Macromolecule #12: Proteasome subunit alpha type-5

• Macromolecule: Name: Proteasome subunit alpha type-5 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 28.649086 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

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 AAESPEEADV EMS

UniProtKB: Proteasome subunit alpha type-5

Macromolecule #13: Proteasome subunit alpha type-6

• Macromolecule: Name: Proteasome subunit alpha type-6 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 25.634 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

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

Macromolecule #14: Probable proteasome subunit alpha type-7

• Macromolecule: Name: Probable proteasome subunit alpha type-7 / type: protein_or_peptide / ID: 14 / Number of copies: 1 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 31.575068 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MTSIGTGYDL SNSVFSPDGR NFQVEYAVKA VENGTTSIGI KCNDGVVFAV EKLITSKLLV PQKNVKIQVV DRHIGCVYSG LIPDGRHLV NRGREEAASF KKLYKTPIPI PAFADRLGQY VQAHTLYNSV RPFGVSTIFG GVDKNGAHLY MLEPSGSYWG Y KGAATGKG RQSAKAELEK LVDHHPEGLS AREAVKQAAK IIYLAHEDNK EKDFELEISW CSLSETNGLH KFVKGDLLQE AI DFAQKEI NGDDDEDEDD SDNVMSSDDE NAPVATNANA TTDQEGDIHL E

UniProtKB: Probable proteasome subunit alpha type-7

Macromolecule #15: 26S proteasome regulatory subunit 7 homolog

• Macromolecule: Name: 26S proteasome regulatory subunit 7 homolog / type: protein_or_peptide / ID: 15 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 52.054891 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MPPKEDWEKY KAPLEDDDKK PDDDKIVPLT EGDIQVLKSY GAAPYAAKLK QTENDLKDIE ARIKEKAGVK ESDTGLAPSH LWDIMGDRQ RLGEEHPLQV ARCTKIIKGN GESDETTTDN NNSGNSNSNS NQQSTDADED DEDAKYVINL KQIAKFVVGL G ERVSPTDI EEGMRVGVDR SKYNIELPLP PRIDPSVTMM TVEEKPDVTY SDVGGCKDQI EKLREVVELP LLSPERFATL GI DPPKGIL LYGPPGTGKT LCARAVANRT DATFIRVIGS ELVQKYVGEG ARMVRELFEM ARTKKACIIF FDEIDAVGGA RFD DGAGGD NEVQRTMLEL ITQLDGFDPR GNIKVMFATN RPNTLDPALL RPGRIDRKVE FSLPDLEGRA NIFRIHSKSM SVER GIRWE LISRLCPNST GAELRSVCTE AGMFAIRARR KVATEKDFLK AVDKVISGYK KFSSTSRYMQ YN

UniProtKB: 26S proteasome regulatory subunit 7 homolog

Macromolecule #16: 26S proteasome regulatory subunit 4 homolog

• Macromolecule: Name: 26S proteasome regulatory subunit 4 homolog / type: protein_or_peptide / ID: 16 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 48.89816 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MGQGVSSGQD KKKKKGSNQK PKYEPPVQSK FGRKKRKGGP ATAEKLPNIY PSTRCKLKLL RMERIKDHLL LEEEFVSNSE ILKPFEKKQ EEEKKQLEEI RGNPLSIGTL EEIIDDDHAI VTSPTMPDYY VSILSFVDKE LLEPGCSVLL HHKTMSIVGV L QDDADPMV SVMKMDKSPT ESYSDIGGLE SQIQEIKESV ELPLTHPELY EEMGIKPPKG VILYGAPGTG KTLLAKAVAN QT SATFLRI VGSELIQKYL GDGPRLCRQI FKVAGENAPS IVFIDEIDAI GTKRYDSNSG GEREIQRTML ELLNQLDGFD DRG DVKVIM ATNKIETLDP ALIRPGRIDR KILFENPDLS TKKKILGIHT SKMNLSEDVN LETLVTTKDD LSGADIQAMC TEAG LLALR ERRMQVTAED FKQAKERVMK NKVEENLEGL YL

UniProtKB: 26S proteasome regulatory subunit 4 homolog

Macromolecule #17: 26S proteasome regulatory subunit 8 homolog

• Macromolecule: Name: 26S proteasome regulatory subunit 8 homolog / type: protein_or_peptide / ID: 17 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 45.342742 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MTAAVTSSNI VLETHESGIK PYFEQKIQET ELKIRSKTEN VRRLEAQRNA LNDKVRFIKD ELRLLQEPGS YVGEVIKIVS DKKVLVKVQ PEGKYIVDVA KDINVKDLKA SQRVCLRSDS YMLHKVLENK ADPLVSLMMV EKVPDSTYDM VGGLTKQIKE I KEVIELPV KHPELFESLG IAQPKGVILY GPPGTGKTLL ARAVAHHTDC KFIRVSGAEL VQKYIGEGSR MVRELFVMAR EH APSIIFM DEIDSIGSTR VEGSGGGDSE VQRTMLELLN QLDGFETSKN IKIIMATNRL DILDPALLRP GRIDRKIEFP PPS VAARAE ILRIHSRKMN LTRGINLRKV AEKMNGCSGA DVKGVCTEAG MYALRERRIH VTQEDFELAV GKVMNKNQET AISV AKLFK

UniProtKB: 26S proteasome regulatory subunit 8 homolog

Macromolecule #18: 26S proteasome regulatory subunit 6B homolog

• Macromolecule: Name: 26S proteasome regulatory subunit 6B homolog / type: protein_or_peptide / ID: 18 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 47.953676 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MEELGIVTPV EKAVEEKPAV KSYASLLAQL NGTVNNNSAL SNVNSDIYFK LKKLEKEYEL LTLQEDYIKD EQRHLKRELK RAQEEVKRI QSVPLVIGQF LEPIDQNTGI VSSTTGMSYV VRILSTLDRE LLKPSMSVAL HRHSNALVDI LPPDSDSSIS V MGENEKPD VTYADVGGLD MQKQEIREAV ELPLVQADLY EQIGIDPPRG VLLYGPPGTG KTMLVKAVAN STKAAFIRVN GS EFVHKYL GEGPRMVRDV FRLARENAPS IIFIDEVDSI ATKRFDAQTG SDREVQRILI ELLTQMDGFD QSTNVKVIMA TNR ADTLDP ALLRPGRLDR KIEFPSLRDR RERRLIFGTI ASKMSLAPEA DLDSLIIRND SLSGAVIAAI MQEAGLRAVR KNRY VILQS DLEEAYATQV KTDNTVDKFD FYK

UniProtKB: 26S proteasome regulatory subunit 6B homolog

Macromolecule #19: 26S proteasome subunit RPT4

• Macromolecule: Name: 26S proteasome subunit RPT4 / type: protein_or_peptide / ID: 19 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 49.480137 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MSEEQDPLLA GLGETSGDNH TQQSHEQQPE QPQETEEHHE EEPSRVDPEQ EAHNKALNQF KRKLLEHRRY DDQLKQRRQN IRDLEKLYD KTENDIKALQ SIGQLIGEVM KELSEEKYIV KASSGPRYIV GVRNSVDRSK LKKGVRVTLD ITTLTIMRIL P RETDPLVY NMTSFEQGEI TFDGIGGLTE QIRELREVIE LPLKNPEIFQ RVGIKPPKGV LLYGPPGTGK TLLAKAVAAT IG ANFIFSP ASGIVDKYIG ESARIIREMF AYAKEHEPCI IFMDEVDAIG GRRFSEGTSA DREIQRTLME LLTQMDGFDN LGQ TKIIMA TNRPDTLDPA LLRPGRLDRK VEIPLPNEAG RLEIFKIHTA KVKKTGEFDF EAAVKMSDGF NGADIRNCAT EAGF FAIRD DRDHINPDDL MKAVRKVAEV KKLEGTIEYQ KL

UniProtKB: 26S proteasome subunit RPT4

Macromolecule #20: 26S proteasome regulatory subunit 6A

• Macromolecule: Name: 26S proteasome regulatory subunit 6A / type: protein_or_peptide / ID: 20 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 48.315727 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MATLEELDAQ TLPGDDELDQ EILNLSTQEL QTRAKLLDNE IRIFRSELQR LSHENNVMLE KIKDNKEKIK NNRQLPYLVA NVVEVMDMN EIEDKENSES TTQGGNVNLD NTAVGKAAVV KTSSRQTVFL PMVGLVDPDK LKPNDLVGVN KDSYLILDTL P SEFDSRVK AMEVDEKPTE TYSDVGGLDK QIEELVEAIV LPMKRADKFK DMGIRAPKGA LMYGPPGTGK TLLARACAAQ TN ATFLKLA APQLVQMYIG EGAKLVRDAF ALAKEKAPTI IFIDELDAIG TKRFDSEKSG DREVQRTMLE LLNQLDGFSS DDR VKVLAA TNRVDVLDPA LLRSGRLDRK IEFPLPSEDS RAQILQIHSR KMTTDDDINW QELARSTDEF NGAQLKAVTV EAGM IALRN GQSSVKHEDF VEGISEVQAR KSKSVSFYA

UniProtKB: 26S proteasome regulatory subunit 6A

Macromolecule #21: Proteasome subunit beta type-7

• Macromolecule: Name: Proteasome subunit beta type-7 / type: protein_or_peptide / ID: 21 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast)
• Molecular weight: Theoretical: 1.789039 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

KWDFAKDIKG YGTQK

Macromolecule #22: Ubiquitin carboxyl-terminal hydrolase RPN11

• Macromolecule: Name: Ubiquitin carboxyl-terminal hydrolase RPN11 / type: protein_or_peptide / ID: 22 / Number of copies: 1 / Enantiomer: LEVO / EC number: ubiquitinyl hydrolase 1
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 34.442281 KDa
• Recombinant expression: Organism: Saccharomyces cerevisiae W303 (yeast)

Sequence

String:

MERLQRLMMN SKVGSADTGR DDTKETVYIS SIALLKMLKH GRAGVPMEVM GLMLGEFVDD YTVNVVDVFA MPQSGTGVSV EAVDDVFQA KMMDMLKQTG RDQMVVGWYH SHPGFGCWLS SVDVNTQKSF EQLNSRAVAV VVDPIQSVKG KVVIDAFRLI D TGALINNL EPRQTTSNTG LLNKANIQAL IHGLNRHYYS LNIDYHKTAK ETKMLMNLHK EQWQSGLKMY DYEEKEESNL AA TKSMVKI AEQYSKRIEE EKELTEEELK TRYVGRQDPK KHLSETADET LENNIVSVLT AGVNSVAIK

UniProtKB: Ubiquitin carboxyl-terminal hydrolase RPN11

Macromolecule #23: Model substrate polypeptide

• Macromolecule: Name: Model substrate polypeptide / type: protein_or_peptide / ID: 23 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 4.068414 KDa
• Recombinant expression: Organism: Escherichia coli BL21 (bacteria)

Sequence

String:

GGKHTFNNEN VSARLGGASI AVQAPAQPPP YSHHHHHH

Macromolecule #24: Ubiquitin-60S ribosomal protein L40

• Macromolecule: Name: Ubiquitin-60S ribosomal protein L40 / type: protein_or_peptide / ID: 24 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c
• Molecular weight: Theoretical: 14.583077 KDa
• Recombinant expression: Organism: Escherichia coli B (bacteria)

Sequence

String:

MQIFVKTLTG KTITLEVESS DTIDNVKSKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGGIIEP SLKALASKY NCDKSVCRKC YARLPPRATN CRKRKCGHTN QLRPKKKLK

UniProtKB: Ubiquitin-ribosomal protein eL40A fusion protein

Macromolecule #25: ADENOSINE-5'-TRIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 25 / Number of copies: 4 / Formula: ATP
• Molecular weight: Theoretical: 507.181 Da

Chemical component information

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

Macromolecule #26: ADENOSINE-5'-DIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 26 / Number of copies: 2 / Formula: ADP
• Molecular weight: Theoretical: 427.201 Da

Chemical component information

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

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 25 mg/mL

Buffer

pH: 7.6
Component:

Concentration	Name
20.0 mM	HEPES
25.0 mM	NaCl
25.0 mM	KCl
10.0 mM	MgCl2
1.0 mM	TCEP
5.0 mM	ATP
0.05 % (w/v)	Nonidet P-40
6.0 mM	ortho-phenanthroline

• Grid: Support film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER; Details: specimens were manually blotted with Whatman #1 filter paper.
• Details: 26S 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

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Details: images were acquired in nanoprobe mode
• Image recording: Film 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/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 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 stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: Camera was operated in counting mode
• Particle selection: Number selected: 579361 ; Details: Particles were selected using the Relion template-based particle picker

Startup model

Type of model: PDB ENTRY
PDB model - PDB ID:

5mp9

• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 48335
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)

Atomic model buiding 1

Initial model

PDB ID:

5mpc

Chain - Source name: PDB / Chain - Initial model type: experimental model

• Refinement: Space: REAL / Protocol: RIGID BODY FIT

Output model

PDB-6ef3:
Yeast 26S proteasome bound to ubiquitinated substrate (4D motor state)