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

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Basic information

Entry
Database: PDB / ID: 8tqm
TitleCryo-EM structure of E3 ubiquitin ligase Doa10 from Saccharomyces cerevisiae
ComponentsERAD-associated E3 ubiquitin-protein ligase DOA10
KeywordsLIGASE / ubiquitin / ERAD / protein quality control / membrane protein
Function / homology
Function and homology information


Doa10p ubiquitin ligase complex / nuclear inner membrane / retrograde protein transport, ER to cytosol / ERAD pathway / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / nuclear envelope / protein ubiquitination / endoplasmic reticulum membrane ...Doa10p ubiquitin ligase complex / nuclear inner membrane / retrograde protein transport, ER to cytosol / ERAD pathway / 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
ERGOSTEROL / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / TRISTEAROYLGLYCEROL / CHOLESTEROL HEMISUCCINATE / ERAD-associated E3 ubiquitin-protein ligase DOA10
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPark, E. / Itskanov, S.I.
Funding support United States, 2items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ERAD-associated E3 ubiquitin-protein ligase DOA10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,5448
Polymers151,6081
Non-polymers4,9357
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 151608.109 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / References: UniProt: P40318
#2: Chemical
ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
#3: Chemical ChemComp-TGL / TRISTEAROYLGLYCEROL / TRIACYLGLYCEROL


Mass: 891.480 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C57H110O6
#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#5: Chemical ChemComp-ERG / ERGOSTEROL


Mass: 396.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H44O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: E3 ubiquitin ligase Doa10 / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.151 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenConc.: 5.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.02% GDN
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 64000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Warp1.07particle selection
2cryoSPARC2.15particle selection
3SerialEMimage acquisition
5cryoSPARC2.15CTF correction
10cryoSPARC2.15initial Euler assignment
11cryoSPARC3final Euler assignment
13cryoSPARC3.0.03D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324019 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingDetails: de novo / Source name: Other / Type: other

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