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- EMDB-17597: cryo-EM structure of Doa10 in MSP1E3D1 -

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Basic information

Entry
Database: EMDB / ID: EMD-17597
Titlecryo-EM structure of Doa10 in MSP1E3D1
Map data
Sample
  • Complex: Doa10 with Ubc6 and sybody in MSP1E3D1
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase DOA10
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE
KeywordsERAD / Doa10 / March6 / TEB4 / retrotranslocation / ubiquitination / Ubc6 / sybody / SQLE / squalenemonooxygenase / LIGASE
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
ERAD-associated E3 ubiquitin-protein ligase DOA10
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsBotsch JJ / Braeuning B / 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 11, 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_17597.png

Downloads & links

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Map

FileDownload / File: emd_17597.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 304 pix.
= 258.765 Å		0.85 Å/pix.
x 304 pix.
= 258.765 Å		0.85 Å/pix.
x 304 pix.
= 258.765 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8512 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.471
Minimum - Maximum-2.471595 - 4.015169
Average (Standard dev.)0.007819354 (±0.08924213)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 258.7648 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17597_msk_1.map
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Half map: #2

Fileemd_17597_half_map_1.map
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Half map: #1

Fileemd_17597_half_map_2.map
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Sample components

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Entire : Doa10 with Ubc6 and sybody in MSP1E3D1

EntireName: Doa10 with Ubc6 and sybody in MSP1E3D1
Components
  • Complex: Doa10 with Ubc6 and sybody in MSP1E3D1
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase DOA10
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

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Supramolecule #1: Doa10 with Ubc6 and sybody in MSP1E3D1

SupramoleculeName: Doa10 with Ubc6 and sybody in MSP1E3D1 / 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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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 / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 151.608109 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
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-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 2 / Number of copies: 3 / Formula: PCW
Molecular weightTheoretical: 787.121 Da
Chemical component information

ChemComp-PCW:
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DOPC, phospholipid*YM

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Macromolecule #3: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

MacromoleculeName: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: PX6
Molecular weightTheoretical: 647.883 Da
Chemical component information

ChemComp-PX6:
1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

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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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 123143
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8pd0:
cryo-EM structure of Doa10 in MSP1E3D1

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