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- EMDB-17610: Doa10 in MSP2N2 -

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Basic information

Entry
Database: EMDB / ID: EMD-17610
TitleDoa10 in MSP2N2
Map data
Sample
  • Complex: Doa10 in MSP2N2
KeywordsERAD / Doa10 / March6 / TEB4 / retrotranslocation / ubiquitination / Ubc6 / sybody / SQLE / squalenemonooxygenase / LIGASE
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.77 Å
AuthorsBotsch JJ / Schulman BA
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2024
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
DepositionJun 12, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17610.png

Downloads & links

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Map

FileDownload / File: emd_17610.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.885 Å
Density
Contour LevelBy AUTHOR: 1.26
Minimum - Maximum-4.4142685 - 6.846909
Average (Standard dev.)-0.0022930822 (±0.14729886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 301.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17610_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Half map: #2

Fileemd_17610_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17610_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Doa10 in MSP2N2

EntireName: Doa10 in MSP2N2
Components
  • Complex: Doa10 in MSP2N2

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Supramolecule #1: Doa10 in MSP2N2

SupramoleculeName: Doa10 in MSP2N2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 150 KDa

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.3000000000000003 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 70.0 e/Å2

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.77 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 188744
FSC plot (resolution estimation)

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