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- EMDB-8637: CryoEM structure of the ERAD-associated E3 ubiquitin-protein liga... -

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Basic information

Entry
Database: EMDB / ID: EMD-8637
TitleCryoEM structure of the ERAD-associated E3 ubiquitin-protein ligase HRD1
Map dataHrd1 dimer map filtered to 4.1A and sharpened with -230 b-factor
Sample
  • Complex: Hrd1 dimer in Hrd1/Hrd3 complex
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase HRD1
KeywordsRetrotranslocon / E3 ligase / ERAD / TRANSFERASE
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 / ERAD pathway / retrograde protein transport, ER to cytosol / protein K48-linked ubiquitination / protein autoubiquitination / : / endoplasmic reticulum unfolded protein response ...Hrd1p ubiquitin ligase ERAD-M complex / Hrd1p ubiquitin ligase complex / Hrd1p ubiquitin ligase ERAD-L complex / fungal-type cell wall organization / ERAD pathway / retrograde protein transport, ER to cytosol / protein K48-linked ubiquitination / protein autoubiquitination / : / endoplasmic reticulum unfolded protein response / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / ubiquitin-dependent protein catabolic process / endoplasmic reticulum membrane / endoplasmic reticulum / identical protein binding / metal ion binding / cytoplasm
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 S288c (yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsSchoebel S / Mi W
Funding support United States, 1 items
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, 2017-
Header (metadata) releaseAug 16, 2017-
Map releaseAug 16, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5v6p
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8637.png

Downloads & links

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Map

FileDownload / File: emd_8637.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHrd1 dimer map filtered to 4.1A and sharpened with -230 b-factor
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 192 pix.
= 259.2 Å		1.35 Å/pix.
x 192 pix.
= 259.2 Å		1.35 Å/pix.
x 192 pix.
= 259.2 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.15739287 - 0.22296952
Average (Standard dev.)-0.000056658606 (±0.008797964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z259.200259.200259.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.1570.223-0.000

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Supplemental data

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Additional map: Hrd1 dimer map without filtering or amplitude modification

Fileemd_8637_additional.map
AnnotationHrd1 dimer map without filtering or amplitude modification
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hrd1 dimer in Hrd1/Hrd3 complex

EntireName: Hrd1 dimer in Hrd1/Hrd3 complex
Components
  • Complex: Hrd1 dimer in Hrd1/Hrd3 complex
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase HRD1

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Supramolecule #1: Hrd1 dimer in Hrd1/Hrd3 complex

SupramoleculeName: Hrd1 dimer in Hrd1/Hrd3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)

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Macromolecule #1: ERAD-associated E3 ubiquitin-protein ligase HRD1

MacromoleculeName: ERAD-associated E3 ubiquitin-protein ligase HRD1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 47.866418 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVPENRRKQL AIFVVVTYLL TFYCVYSATK TSVSFLQVTL KLNEGFNLMV LSIFILLNST LLWQLLTKLL FGELRLIEHE HIFERLPFT IINTLFMSSL FHERYFFTVA FFGLLLLYLK VFHWILKDRL EALLQSINDS TTMKTLIFSR FSFNLVLLAV V DYQIITRC ...String:
MVPENRRKQL AIFVVVTYLL TFYCVYSATK TSVSFLQVTL KLNEGFNLMV LSIFILLNST LLWQLLTKLL FGELRLIEHE HIFERLPFT IINTLFMSSL FHERYFFTVA FFGLLLLYLK VFHWILKDRL EALLQSINDS TTMKTLIFSR FSFNLVLLAV V DYQIITRC ISSIYTNQKS DIESTSLYLI QVMEFTMLLI DLLNLFLQTC LNFWEFYRSQ QSLSNENNHI VHGDPTDENT VE SDQSQPV LNDDDDDDDD DRQFTGLEGK FMYEKAIDVF TRFLKTALHL SMLIPFRMPM MLLKDVVWDI LALYQSGTSL WKI WRNNKQ LDDTLVTVTV EQLQNSANDD NICIICMDEL IHSPNQQTWK NKNKKPKRLP CGHILHLSCL KNWMERSQTC PICR LPVFD EK

UniProtKB: ERAD-associated E3 ubiquitin-protein ligase HRD1

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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

Concentration0.8 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 82.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: spider projection matching starting with a Gaussian density
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: GeRelion (ver. 1.0) / Number images used: 93609
FSC plot (resolution estimation)

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