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- PDB-6vk3: CryoEM structure of Hrd3/Yos9 complex -

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Basic information

Entry
Database: PDB / ID: 6vk3
TitleCryoEM structure of Hrd3/Yos9 complex
Components
  • Hrd3
  • Protein OS-9 homolog
KeywordsPROTEIN TRANSPORT / retro-translocation / ERAD / protein degradation / ubiquitination / glycan recognition
Function / homology
Function and homology information


ubiquitin-dependent glycoprotein ERAD pathway / Hrd1p ubiquitin ligase ERAD-M complex / detection of unfolded protein / luminal surveillance complex / Hrd1p ubiquitin ligase complex / Hrd1p ubiquitin ligase ERAD-L complex / oligosaccharide binding / negative regulation of protein autoubiquitination / retrograde protein transport, ER to cytosol / endoplasmic reticulum unfolded protein response ...ubiquitin-dependent glycoprotein ERAD pathway / Hrd1p ubiquitin ligase ERAD-M complex / detection of unfolded protein / luminal surveillance complex / Hrd1p ubiquitin ligase complex / Hrd1p ubiquitin ligase ERAD-L complex / oligosaccharide binding / negative regulation of protein autoubiquitination / retrograde protein transport, ER to cytosol / endoplasmic reticulum unfolded protein response / ERAD pathway / endoplasmic reticulum lumen / endoplasmic reticulum membrane / endoplasmic reticulum / identical protein binding
Similarity search - Function
Yos9, dimerzation domain / Yos9 dimerzation domain / Protein OS9-like domain / Protein OS-9-like / Glucosidase II beta subunit-like protein / : / Sel1 repeat / Sel1-like repeat / Sel1-like repeats. / Mannose-6-phosphate receptor binding domain superfamily ...Yos9, dimerzation domain / Yos9 dimerzation domain / Protein OS9-like domain / Protein OS-9-like / Glucosidase II beta subunit-like protein / : / Sel1 repeat / Sel1-like repeat / Sel1-like repeats. / Mannose-6-phosphate receptor binding domain superfamily / MRH domain / MRH domain profile. / Endoplasmic reticulum targeting sequence. / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
ERAD-associated E3 ubiquitin-protein ligase component HRD3 / Protein OS-9 homolog
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsWu, X. / Rapoport, T.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM052586 United States
CitationJournal: Science / Year: 2020
Title: Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex.
Authors: Xudong Wu / Marc Siggel / Sergey Ovchinnikov / Wei Mi / Vladimir Svetlov / Evgeny Nudler / Maofu Liao / Gerhard Hummer / Tom A Rapoport /
Abstract: Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD- ...Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo-electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two "half-channels" with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.
History
DepositionJan 18, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Hrd3
B: Protein OS-9 homolog


Theoretical massNumber of molelcules
Total (without water)145,6392
Polymers145,6392
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Hrd3


Mass: 84328.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: Hrd3 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q05787*PLUS
#2: Protein Protein OS-9 homolog


Mass: 61311.285 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: YOS9 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q99220
Has protein modificationY
Sequence detailsThe full sequence of poly-UNK region in HRD3 is PDIGSPFIAQVNGVQMTLQIEPMGRFAFNGNDGNINGDEDDE, UniProt ...The full sequence of poly-UNK region in HRD3 is PDIGSPFIAQVNGVQMTLQIEPMGRFAFNGNDGNINGDEDDE, UniProt reference ID is Q05787.

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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: a complex of Hrd1/Hrd3/Yos9 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
225 mMHEPESC8H18N2O4S1
30.06 %digitoninC56H92O291
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: OTHER
Image recordingElectron dose: 44.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
4GctfCTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99298 / Symmetry type: POINT
RefinementHighest resolution: 3.7 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0067959
ELECTRON MICROSCOPYf_angle_d0.92310768
ELECTRON MICROSCOPYf_dihedral_angle_d21.4252924
ELECTRON MICROSCOPYf_chiral_restr0.1641160
ELECTRON MICROSCOPYf_plane_restr0.0061383

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