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- PDB-6vk0: CryoEM structure of Hrd1-Usa1/Der1/Hrd3 of the flipped topology -

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Basic information

Entry
Database: PDB / ID: 6vk0
TitleCryoEM structure of Hrd1-Usa1/Der1/Hrd3 of the flipped topology
Components
  • Degradation in the endoplasmic reticulum protein 1
  • ERAD-associated E3 ubiquitin-protein ligase HRD1
  • ERAD-associated E3 ubiquitin-protein ligase component HRD3
  • U1 SNP1-associating protein 1
KeywordsPROTEIN TRANSPORT / retro-translocation / ERAD / protein degradation / ubiquitination
Function / homology
Function and homology information


Hrd1p ubiquitin ligase ERAD-M complex / luminal surveillance complex / misfolded protein transport / negative regulation of protein autoubiquitination / Hrd1p ubiquitin ligase ERAD-L complex / positive regulation of protein autoubiquitination / fungal-type cell wall organization / retrograde protein transport, ER to cytosol / protein quality control for misfolded or incompletely synthesized proteins / misfolded protein binding ...Hrd1p ubiquitin ligase ERAD-M complex / luminal surveillance complex / misfolded protein transport / negative regulation of protein autoubiquitination / Hrd1p ubiquitin ligase ERAD-L complex / positive regulation of protein autoubiquitination / fungal-type cell wall organization / retrograde protein transport, ER to cytosol / protein quality control for misfolded or incompletely synthesized proteins / misfolded protein binding / ubiquitin-specific protease binding / protein K48-linked ubiquitination / integral component of endoplasmic reticulum membrane / protein autoubiquitination / molecular adaptor activity / endoplasmic reticulum unfolded protein response / ubiquitin-dependent ERAD pathway / RING-type E3 ubiquitin transferase / mRNA splicing, via spliceosome / ubiquitin protein ligase activity / ubiquitin-protein transferase activity / ubiquitin-dependent protein catabolic process / protein ubiquitination / endoplasmic reticulum membrane / endoplasmic reticulum / integral component of membrane / identical protein binding / metal ion binding
Sel1-like repeat / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain / Tetratricopeptide-like helical domain superfamily / Derlin / Ubiquitin-like domain superfamily / E3 ubiquitin-protein ligase synoviolin/Hrd1 / Zinc finger, RING-type
Degradation in the endoplasmic reticulum protein 1 / U1 SNP1-associating protein 1 / ERAD-associated E3 ubiquitin-protein ligase component HRD3 / ERAD-associated E3 ubiquitin-protein ligase HRD1
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 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.
Validation Report
SummaryFull reportAbout validation report
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

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Structure visualization

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Assembly

Deposited unit
D: U1 SNP1-associating protein 1
C: Degradation in the endoplasmic reticulum protein 1
A: ERAD-associated E3 ubiquitin-protein ligase component HRD3
B: ERAD-associated E3 ubiquitin-protein ligase HRD1


Theoretical massNumber of molelcules
Total (without water)208,2344
Polymers208,2344
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein U1 SNP1-associating protein 1


Mass: 39944.301 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: USA1 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q03714
#2: Protein Degradation in the endoplasmic reticulum protein 1


Mass: 24411.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: DER1 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P38307
#3: Protein ERAD-associated E3 ubiquitin-protein ligase component HRD3 / HMG-CoA reductase degradation protein 3


Mass: 88239.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: HRD3 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q05787
#4: Protein ERAD-associated E3 ubiquitin-protein ligase HRD1 / HMG-CoA reductase degradation protein 1 / RING-type E3 ubiquitin transferase HRD1


Mass: 55638.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: HRD1, DER3 / Production host: Saccharomyces cerevisiae (baker'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: complex of Hrd1-Usa1/Der1/Hrd3 of the flipped topology
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1250 mMsodium chlorideNaClSodium chloride1
225 mMHEPESC8H18N2O4S1
30.06 %digitoninC56H92O291
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 54.8 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k 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
11RELION3classification
13PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 252744 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0079921
ELECTRON MICROSCOPYf_angle_d0.81813449
ELECTRON MICROSCOPYf_dihedral_angle_d18.9373602
ELECTRON MICROSCOPYf_chiral_restr0.0471515
ELECTRON MICROSCOPYf_plane_restr0.0051651

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