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- EMDB-21221: CryoEM map of Hrd1-Usa1/Der1/Hrd3 complex of the expected topology -

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Basic information

Entry
Database: EMDB / ID: EMD-21221
TitleCryoEM map of Hrd1-Usa1/Der1/Hrd3 complex of the expected topology
Map data
Samplecomplex of Hrd1-Usa1/Der1/Hrd3 in the expected topology
  • Degradation in the endoplasmic reticulum protein 1
  • (ERAD-associated E3 ubiquitin-protein ligase ...) x 2
  • U1 SNP1-associating protein 1
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsWu X / Rapoport TA
Funding support United States, 1 items
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 ReportSummary, Full report, XML, About validation report
History
DepositionJan 18, 2020-
Header (metadata) releaseApr 29, 2020-
Map releaseApr 29, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6vjz
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21221.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 160 pix.
= 216. Å
1.35 Å/pix.
x 160 pix.
= 216. Å
1.35 Å/pix.
x 160 pix.
= 216. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.027
Minimum - Maximum-0.10065114 - 0.14309451
Average (Standard dev.)0.00003767069 (±0.0061342116)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 216.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z216.000216.000216.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1010.1430.000

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Supplemental data

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Additional map: unsharpened map

Fileemd_21221_additional.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire complex of Hrd1-Usa1/Der1/Hrd3 in the expected topology

EntireName: complex of Hrd1-Usa1/Der1/Hrd3 in the expected topology
Number of components: 5

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Component #1: protein, complex of Hrd1-Usa1/Der1/Hrd3 in the expected topology

ProteinName: complex of Hrd1-Usa1/Der1/Hrd3 in the expected topology
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Degradation in the endoplasmic reticulum protein 1

ProteinName: Degradation in the endoplasmic reticulum protein 1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 24.411715 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, ERAD-associated E3 ubiquitin-protein ligase HRD1

ProteinName: ERAD-associated E3 ubiquitin-protein ligase HRD1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 55.638773 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #4: protein, ERAD-associated E3 ubiquitin-protein ligase component HRD3

ProteinName: ERAD-associated E3 ubiquitin-protein ligase component HRD3
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 88.239102 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #5: protein, U1 SNP1-associating protein 1

ProteinName: U1 SNP1-associating protein 1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 39.944301 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 5 mg/mL / pH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 54.8 e/Å2 / Illumination mode: OTHER
LensImaging mode: OTHER
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 172291
3D reconstructionSoftware: RELION / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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