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Open data
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Basic information
Entry | Database: PDB / ID: 8pd0 | ||||||
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Title | cryo-EM structure of Doa10 in MSP1E3D1 | ||||||
![]() | ERAD-associated E3 ubiquitin-protein ligase DOA10 | ||||||
![]() | LIGASE / ERAD / Doa10 / March6 / TEB4 / retrotranslocation / ubiquitination / Ubc6 / sybody / SQLE / squalenemonooxygenase | ||||||
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: 3.58 Å | ||||||
![]() | Botsch, J.J. / Braeuning, B. / Schulman, B.A. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE. Authors: J Josephine Botsch / Roswitha Junker / Michèle Sorgenfrei / Patricia P Ogger / Luca Stier / Susanne von Gronau / Peter J Murray / Markus A Seeger / Brenda A Schulman / Bastian Bräuning / ![]() ![]() Abstract: Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in ...Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of Doa10/MARCH6 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, and gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Here, by assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, we reveal crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected Doa10/MARCH6 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 172 KB | Display | ![]() |
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PDB format | ![]() | 121.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.3 MB | Display | ![]() |
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Full document | ![]() | 1.3 MB | Display | |
Data in XML | ![]() | 39.5 KB | Display | |
Data in CIF | ![]() | 57.9 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 17597MC ![]() 8pdaC 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 genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: SSM4, DOA10, YIL030C, YI3299.01C, YI9905.18C / Production host: ![]() References: UniProt: P40318, RING-type E3 ubiquitin transferase | ||||
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#2: Chemical | #3: Chemical | ChemComp-PX6 / | 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: Doa10 with Ubc6 and sybody in MSP1E3D1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.15 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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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 defocus max: 2800 nm / Nominal defocus min: 700 nm |
Image recording | Electron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
EM software | Name: PHENIX / Version: 1.20.1_4487: / Category: model refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123143 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model | ||||||||||||||||||||||||
Refine LS restraints |
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