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- PDB-8tqm: Cryo-EM structure of E3 ubiquitin ligase Doa10 from Saccharomyces... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8tqm | |||||||||
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Title | Cryo-EM structure of E3 ubiquitin ligase Doa10 from Saccharomyces cerevisiae | |||||||||
![]() | ERAD-associated E3 ubiquitin-protein ligase DOA10 | |||||||||
![]() | LIGASE / ubiquitin / ERAD / protein quality control / membrane protein | |||||||||
Function / homology | ![]() 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 | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
![]() | Park, E. / Itskanov, S.I. | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 191.8 KB | Display | ![]() |
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PDB format | ![]() | 145.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.4 MB | Display | ![]() |
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Full document | ![]() | 1.5 MB | Display | |
Data in XML | ![]() | 45.8 KB | Display | |
Data in CIF | ![]() | 64.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 41508MC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 151608.109 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() | ||||||||
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#2: Chemical | ChemComp-PC1 / #3: Chemical | ChemComp-TGL / | #4: Chemical | ChemComp-Y01 / | #5: Chemical | ChemComp-ERG / | Has ligand of interest | N | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: E3 ubiquitin ligase Doa10 / Type: COMPLEX / Entity ID: #1 / Source: NATURAL |
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Molecular weight | Value: 0.151 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 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 support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 64000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 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 building | Protocol: AB INITIO MODEL | ||||||||||||||||||||||||||||||||||||
Atomic model building | Details: de novo / Source name: Other / Type: other |