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- EMDB-25750: Structure of the peroxisomal retro-translocon formed by a heterot... -

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Basic information

Entry
Database: EMDB / ID: EMD-25750
TitleStructure of the peroxisomal retro-translocon formed by a heterotrimeric ubiquitin ligase complex
Map dataStructure of the peroxisomal retro-translocon formed by a heterotrimeric ubiquitin ligase complex
Sample
  • Complex: Peroxisomal heterotrimeric ubiquitin ligase complex bound with Fab
    • Complex: Peroxin12, Peroxin2, Peroxin10
      • Protein or peptide: Peroxin-12
      • Protein or peptide: Peroxin-2
      • Protein or peptide: Peroxin-10
    • Complex: Fab heavy chain, Fab light chain
      • Protein or peptide: Fab heavy chainFragment antigen-binding
      • Protein or peptide: Fab light chainFragment antigen-binding
  • Ligand: ZINC ION
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • Ligand: CHOLESTEROL
Keywordsperoxisome / retro-translocon / ubiquitin ligase / TRANSLOCASE
Function / homology
Function and homology information


: / protein import into peroxisome matrix / : / peroxisome / zinc ion binding / metal ion binding
Similarity search - Function
Pex, N-terminal / Peroxisome assembly protein 12 / Pex2 / Pex12 amino terminal region / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
RING-type domain-containing protein / Pex2_Pex12 domain-containing protein / Pex2_Pex12 domain-containing protein
Similarity search - Component
Biological speciesThermothelomyces thermophilus ATCC 42464 (fungus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsPeiqiang F / Tom R
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2022
Title: A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.
Authors: Peiqiang Feng / Xudong Wu / Satchal K Erramilli / Joao A Paulo / Pawel Knejski / Steven P Gygi / Anthony A Kossiakoff / Tom A Rapoport /
Abstract: Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which ...Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12). Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
History
DepositionDec 17, 2021-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25750.png

Downloads & links

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Map

FileDownload / File: emd_25750.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the peroxisomal retro-translocon formed by a heterotrimeric ubiquitin ligase complex
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.14288855 - 0.27496657
Average (Standard dev.)0.00019035877 (±0.006544081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 275.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Peroxisomal heterotrimeric ubiquitin ligase complex bound with Fab

EntireName: Peroxisomal heterotrimeric ubiquitin ligase complex bound with Fab
Components
  • Complex: Peroxisomal heterotrimeric ubiquitin ligase complex bound with Fab
    • Complex: Peroxin12, Peroxin2, Peroxin10
      • Protein or peptide: Peroxin-12
      • Protein or peptide: Peroxin-2
      • Protein or peptide: Peroxin-10
    • Complex: Fab heavy chain, Fab light chain
      • Protein or peptide: Fab heavy chainFragment antigen-binding
      • Protein or peptide: Fab light chainFragment antigen-binding
  • Ligand: ZINC ION
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • Ligand: CHOLESTEROL

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Supramolecule #1: Peroxisomal heterotrimeric ubiquitin ligase complex bound with Fab

SupramoleculeName: Peroxisomal heterotrimeric ubiquitin ligase complex bound with Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: Peroxin12, Peroxin2, Peroxin10

SupramoleculeName: Peroxin12, Peroxin2, Peroxin10 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Thermothelomyces thermophilus ATCC 42464 (fungus)

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Supramolecule #3: Fab heavy chain, Fab light chain

SupramoleculeName: Fab heavy chain, Fab light chain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Peroxin-12

MacromoleculeName: Peroxin-12 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermothelomyces thermophilus ATCC 42464 (fungus) / Strain: ATCC 42464 / BCRC 31852 / DSM 1799
Molecular weightTheoretical: 49.369703 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MEFIPALQAR IDEHKPSLFE LLSEQQLAAL LPPTVRYLLT VLTQRYPRYL LRVLNSFDEL YALAALVVER HYLRTRGGSF TEHFYGLKR ERALAAEIPR ASAAAPGAVR DALRLRPADV WRNLLVIVGV PYLKRKLDEA HEADAPRAMM GAAYNRPPLP G APWRERVA ...String:
MEFIPALQAR IDEHKPSLFE LLSEQQLAAL LPPTVRYLLT VLTQRYPRYL LRVLNSFDEL YALAALVVER HYLRTRGGSF TEHFYGLKR ERALAAEIPR ASAAAPGAVR DALRLRPADV WRNLLVIVGV PYLKRKLDEA HEADAPRAMM GAAYNRPPLP G APWRERVA FWWRCFLRRV YPAVNAAYYL SILAFNLAYL FDNSKYHSPF LCLIGTRVRR MSAADYRAIE ALEERAAKRR GG RSVAARM LGGLSLVLPT SIFALKFLEW WYASDFAKQL SRKAAESLDL PPPVVAGLPT GAGGGGGSEK AQQPRREKEL GKE KDEEVE EEEEEVSEEE KERRAIERAP VSASSLLPIY TVPPPENSDQ CPICEGEITT AAACQTGIVY CYPCIHKWLT GTHP RQEKF MAERAGKWES GEGRCAVTGR RVLGGTEGLR RIMV

UniProtKB: Pex2_Pex12 domain-containing protein

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Macromolecule #2: Peroxin-2

MacromoleculeName: Peroxin-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermothelomyces thermophilus ATCC 42464 (fungus) / Strain: ATCC 42464 / BCRC 31852 / DSM 1799
Molecular weightTheoretical: 38.493715 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: TRPAFRVGQV DAELLDEELV ELLRGQVREA LRYVGGGGGG GGGGGGGGVG SGVAQDWEAE ISLALRAVLF KLTVWDHDAT YGAALQNLK YTDARRDGPA LAPPSRWQKA LYGLVTVGGR YLWAKWEDWL LEQDDGFEGP SPRVKRLARW TSSLSTLHAS A ALVSFLVF ...String:
TRPAFRVGQV DAELLDEELV ELLRGQVREA LRYVGGGGGG GGGGGGGGVG SGVAQDWEAE ISLALRAVLF KLTVWDHDAT YGAALQNLK YTDARRDGPA LAPPSRWQKA LYGLVTVGGR YLWAKWEDWL LEQDDGFEGP SPRVKRLARW TSSLSTLHAS A ALVSFLVF LLHGRYRTLL DRLLRMRLAP PTSQVSREVS FEYLNRQLVW HAFTEFLLFV LPLVGINRWR RWLARTWRRT KK IMTADAD GGAGDKKGEY SFLPERTCAI CYRDQNSASS ETELLAAASG GVVGSAQTDI TNPYEAIPCG CTYCFVCLAT RIE REEGEG WPCLRCGELI KECKPWNGDV L

UniProtKB: Pex2_Pex12 domain-containing protein

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Macromolecule #3: Peroxin-10

MacromoleculeName: Peroxin-10 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermothelomyces thermophilus ATCC 42464 (fungus) / Strain: ATCC 42464 / BCRC 31852 / DSM 1799
Molecular weightTheoretical: 48.952234 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MATQPPPARP PPPLTSSPYP YAAAPDIIRA HQKDAYFQGV LANRLSDLHR RLRGARSAHA WAAETRTFAA ALYLCLTTLL GNRTLGEEY CDLVQVEEAP SKLFASSSSK AADDHIYENG LGGGGDGGPL LPSLPRRAGY ILTAIVLPHL ASRALPSVRS A IRKRLQSR ...String:
MATQPPPARP PPPLTSSPYP YAAAPDIIRA HQKDAYFQGV LANRLSDLHR RLRGARSAHA WAAETRTFAA ALYLCLTTLL GNRTLGEEY CDLVQVEEAP SKLFASSSSK AADDHIYENG LGGGGDGGPL LPSLPRRAGY ILTAIVLPHL ASRALPSVRS A IRKRLQSR LATLSRRRQQ TGTKSGSGRG GRGGGGGITE YRVLRYLLTH LTPLTSGAHF RAATLAVFYF TGAYYELSKW VW GLRYVFT TRAGRVVDDD HNRHHHSPQH GGGNGGRAGY EVLGVLLVVQ MAVRAWLHVR EQLSSGSVAG GGGEEEEDGE DGF RERTAF GPGTNVDVSL DEHAFTSNNE LLGGGGGGGG SSSQRSLGEI GAMAHTPVLK AGRARYDLGT SDKVMGWIKG AQQR KCTLC LEELKDPAAT QCGHVFCWAC IGDWVREKPE CPLCRREAMV QHILPLRAA

UniProtKB: RING-type domain-containing protein

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Macromolecule #4: Fab heavy chain

MacromoleculeName: Fab heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.518806 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
EVQLVESGGG LVQPGGSLRL SCAASGFNLY SSSIHWVRQA PGKGLEWVAY IYSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARG YYSWYKASWA LADYW

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Macromolecule #5: Fab light chain

MacromoleculeName: Fab light chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.135375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QGSSLGLLTF GQGTKV

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

MacromoleculeName: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / type: ligand / ID: 7 / Number of copies: 15 / Formula: LBN
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-LBN:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / phospholipid*YM / POPC

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Macromolecule #8: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 8 / Number of copies: 5 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK I
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 121644

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