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- PDB-5v6p: CryoEM structure of the ERAD-associated E3 ubiquitin-protein liga... -

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Entry
Database: PDB / ID: 5v6p
TitleCryoEM structure of the ERAD-associated E3 ubiquitin-protein ligase HRD1
ComponentsERAD-associated E3 ubiquitin-protein ligase HRD1
KeywordsTRANSFERASE / Retrotranslocon / E3 ligase / ERAD
Function / homology
Function and homology information


Hrd1p ubiquitin ligase ERAD-M complex / Hrd1p ubiquitin ligase complex / Hrd1p ubiquitin ligase ERAD-L complex / fungal-type cell wall organization / retrograde protein transport, ER to cytosol / protein K48-linked ubiquitination / endoplasmic reticulum unfolded protein response / protein autoubiquitination / ERAD pathway / RING-type E3 ubiquitin transferase ...Hrd1p ubiquitin ligase ERAD-M complex / Hrd1p ubiquitin ligase complex / Hrd1p ubiquitin ligase ERAD-L complex / fungal-type cell wall organization / retrograde protein transport, ER to cytosol / protein K48-linked ubiquitination / endoplasmic reticulum unfolded protein response / protein autoubiquitination / ERAD pathway / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / ubiquitin-dependent protein catabolic process / endoplasmic reticulum membrane / endoplasmic reticulum / zinc ion binding / identical protein binding
Similarity search - Function
: / Ring finger domain / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
ERAD-associated E3 ubiquitin-protein ligase HRD1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsSchoebel, S. / Mi, W. / Stein, A. / Rapoport, T.A. / Liao, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM052586 United States
CitationJournal: Nature / Year: 2017
Title: Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3.
Authors: Stefan Schoebel / Wei Mi / Alexander Stein / Sergey Ovchinnikov / Ryan Pavlovicz / Frank DiMaio / David Baker / Melissa G Chambers / Huayou Su / Dongsheng Li / Tom A Rapoport / Maofu Liao /
Abstract: Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome-a ...Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome-a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain. Hrd1 is the crucial membrane component for retro-translocation, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction-from the cytosol into or across membranes. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.
History
DepositionMar 17, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 16, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Author supporting evidence / Database references / Category: citation / citation_author / pdbx_audit_support
Item: _citation.pdbx_database_id_PubMed / _citation.title / _pdbx_audit_support.funding_organization
Revision 1.2Aug 30, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.4Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: ERAD-associated E3 ubiquitin-protein ligase HRD1
B: ERAD-associated E3 ubiquitin-protein ligase HRD1


Theoretical massNumber of molelcules
Total (without water)95,7332
Polymers95,7332
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3610 Å2
ΔGint-41 kcal/mol
Surface area29610 Å2
MethodPISA

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Components

#1: Protein ERAD-associated E3 ubiquitin-protein ligase HRD1 / HMG-CoA reductase degradation protein 1 / RING-type E3 ubiquitin transferase HRD1


Mass: 47866.418 Da / Num. of mol.: 2 / Fragment: UNP residues 1-407
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: HRD1, DER3, YOL013C / Plasmid: pRS426 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: Q08109, RING-type E3 ubiquitin transferase

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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: Hrd1 dimer in Hrd1/Hrd3 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Source (recombinant)Organism: Saccharomyces (fungus)
Buffer solutionpH: 7.5
SpecimenConc.: 0.8 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 82 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: GeRelion / Version: 1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93609 / Symmetry type: POINT

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