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- EMDB-41508: Cryo-EM structure of E3 ubiquitin ligase Doa10 from Saccharomyces... -

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Basic information

Entry
Database: EMDB / ID: EMD-41508
TitleCryo-EM structure of E3 ubiquitin ligase Doa10 from Saccharomyces cerevisiae
Map dataSharpened map
Sample
  • Complex: E3 ubiquitin ligase Doa10
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase DOA10
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: TRISTEAROYLGLYCEROLStearin
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: ERGOSTEROL
Keywordsubiquitin / ERAD / protein quality control / membrane protein / LIGASE
Function / homology
Function and homology information


Doa10p ubiquitin ligase complex / : / nuclear inner membrane / retrograde protein transport, ER to cytosol / : / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / nuclear envelope / protein ubiquitination ...Doa10p ubiquitin ligase complex / : / nuclear inner membrane / retrograde protein transport, ER to cytosol / : / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / nuclear envelope / protein ubiquitination / endoplasmic reticulum membrane / endoplasmic reticulum / zinc ion binding / membrane
Similarity search - Function
RING-variant domain / Zinc finger RING-CH-type profile. / Zinc finger, RING-CH-type / The RING-variant domain is a C4HC3 zinc-finger like motif found in a number of cellular and viral proteins. Some of these proteins have been shown both in vivo and in vitro to have ubiquitin E3 ligase activity. / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
ERAD-associated E3 ubiquitin-protein ligase DOA10
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae BY4741 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPark E / Itskanov SI
Funding support United States, 2 items
OrganizationGrant numberCountry
The Vallee Foundation Inc. United States
The Pew Charitable Trusts United States
CitationJournal: Nat Commun / Year: 2024
Title: Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.
Authors: Kevin Wu / Samuel Itskanov / Diane L Lynch / Yuanyuan Chen / Aasha Turner / James C Gumbart / Eunyong Park /
Abstract: Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. ...Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we define the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its model substrates. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrate's degron peptide into the lateral tunnel is required for efficient polyubiquitination. The N- and C-terminal membrane domains of Doa10 seem to form fence-like features to restrict polyubiquitination to those proteins that can access the central cavity and lateral tunnel. Our study reveals how extended hydrophobic sequences at the termini of substrate proteins are recognized by Doa10 as a signal for quality control.
History
DepositionAug 7, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41508.png

Downloads & links

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Map

FileDownload / File: emd_41508.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.489498 - 2.3612244
Average (Standard dev.)0.001718179 (±0.03157131)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 291.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_41508_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41508_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41508_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E3 ubiquitin ligase Doa10

EntireName: E3 ubiquitin ligase Doa10
Components
  • Complex: E3 ubiquitin ligase Doa10
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase DOA10
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: TRISTEAROYLGLYCEROLStearin
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: ERGOSTEROL

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Supramolecule #1: E3 ubiquitin ligase Doa10

SupramoleculeName: E3 ubiquitin ligase Doa10 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 151 KDa

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Macromolecule #1: ERAD-associated E3 ubiquitin-protein ligase DOA10

MacromoleculeName: ERAD-associated E3 ubiquitin-protein ligase DOA10 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast)
Molecular weightTheoretical: 151.608109 KDa
SequenceString: MDVDSDVNVS RLRDELHKVA NEETDTATFN DDAPSGATCR ICRGEATEDN PLFHPCKCRG SIKYMHESCL LEWVASKNID ISKPGADVK CDICHYPIQF KTIYAENMPE KIPFSLLLSK SILTFFEKAR LALTIGLAAV LYIIGVPLVW NMFGKLYTMM L DGSSPYPG ...String:
MDVDSDVNVS RLRDELHKVA NEETDTATFN DDAPSGATCR ICRGEATEDN PLFHPCKCRG SIKYMHESCL LEWVASKNID ISKPGADVK CDICHYPIQF KTIYAENMPE KIPFSLLLSK SILTFFEKAR LALTIGLAAV LYIIGVPLVW NMFGKLYTMM L DGSSPYPG DFLKSLIYGY DQSATPELTT RAIFYQLLQN HSFTSLQFIM IVILHIALYF QYDMIVREDV FSKMVFHKIG PR LSPKDLK SRLKERFPMM DDRMVEYLAR EMRAHDENRQ EQGHDRLNMP AAAADNNNNV INPRNDNVPP QDPNDHRNFE NLR HVDELD HDEATEEHEN NDSDNSLPSG DDSSRILPGS SSDNEEDEEA EGQQQQQQPE EEADYRDHIE PNPIDMWANR RAQN EFDDL IAAQQNAINR PNAPVFIPPP AQNRAGNVDQ DEQDFGAAVG VPPAQANPDD QGQGPLVINL KLKLLNVIAY FIIAV VFTA IYLAISYLFP TFIGFGLLKI YFGIFKVILR GLCHLYYLSG AHIAYNGLTK LVPKVDVAMS WISDHLIHDI IYLYNG YTE NTMKHSIFIR ALPALTTYLT SVSIVCASSN LVSRGYGREN GMSNPTRRLI FQILFALKCT FKVFTLFFIE LAGFPIL AG VMLDFSLFCP ILASNSRMLW VPSICAIWPP FSLFVYWTIG TLYMYWFAKY IGMIRKNIIR PGVLFFIRSP EDPNIKIL H DSLIHPMSIQ LSRLCLSMFI YAIFIVLGFG FHTRIFFPFM LKSNLLSVPE AYKPTSIISW KFNTILLTLY FTKRILESS SYVKPLLERY WKTIFKLCSR KLRLSSFILG KDTPTERGHI VYRNLFYKYI AAKNAEWSNQ ELFTKPKTLE QAEELFGQVR DVHAYFVPD GVLMRVPSSD IVSRNYVQTM FVPVTKDDKL LKPLDLERIK ERNKRAAGEF GYLDEQNTEY DQYYIVYVPP D FRLRYMTL LGLVWLFASI LMLGVTFISQ ALINFVCSFG FLPVVKLLLG ERNKVYVAWK ELSDISYSYL NIYYVCVGSV CL SKIAKDI LHFTEGQNTL DEHAVDENEV EEVEHDIPER DINNAPVNNI NNVEEGQGIF MAIFNSIFDS MLVKYNLMVF IAI MIAVIR TMVSWVVLTD GILACYNYLT IRVFGNSSYT IGNSKWFKYD ESLLFVVWII SSMVNFGTGY KSLKLFFRNR NTSK LNFLK TMALELFKQG FLHMVIYVLP IIILSLVFLR DVSTKQIIDI SHGSRSFTLS LNESFPTWTR MQDIYFGLLI ALESF TFFF QATVLFIQWF KSTVQNVKDE VYTKGRALEN LPDES

UniProtKB: ERAD-associated E3 ubiquitin-protein ligase DOA10

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Macromolecule #2: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 2 / Number of copies: 4 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM / Phosphatidylcholine

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Macromolecule #3: TRISTEAROYLGLYCEROL

MacromoleculeName: TRISTEAROYLGLYCEROL / type: ligand / ID: 3 / Number of copies: 1 / Formula: TGL
Molecular weightTheoretical: 891.48 Da
Chemical component information

ChemComp-TGL:
TRISTEAROYLGLYCEROL / Stearin

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #5: ERGOSTEROL

MacromoleculeName: ERGOSTEROL / type: ligand / ID: 5 / Number of copies: 1 / Formula: ERG
Molecular weightTheoretical: 396.648 Da
Chemical component information

ChemComp-ERG:
ERGOSTEROL / Ergosterol

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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

Concentration5.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.02% GDN

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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: 2.4 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.0) / Number images used: 324019
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: other / Details: de novo
RefinementProtocol: AB INITIO MODEL
Output model

PDB-8tqm:
Cryo-EM structure of E3 ubiquitin ligase Doa10 from Saccharomyces cerevisiae

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