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- EMDB-8638: Cryo-EM map of the ERAD components Hrd1/Hrd3 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-8638
TitleCryo-EM map of the ERAD components Hrd1/Hrd3 dimer
Map datamap of Hrd1/Hrd3 dimer filtering to 4.7A and applied with -250 bfactor
Sample
  • Complex: Hrd1/Hrd3 dimer
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase HRD1
  • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase component HRD3
Function / homology
Function and homology information


Hrd1p ubiquitin ligase ERAD-M complex / detection of unfolded protein / luminal surveillance complex / Hrd1p ubiquitin ligase complex / ubiquitin-dependent glycoprotein ERAD pathway / Hrd1p ubiquitin ligase ERAD-L complex / negative regulation of protein autoubiquitination / retrograde protein transport, ER to cytosol / : / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Sel1 repeat / Sel1-like repeat / Sel1-like repeats. / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
ERAD-associated E3 ubiquitin-protein ligase component HRD3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288c (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsSchoebel S / Mi W / Stein A / Rapoport TA / Liao M
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) releaseApr 26, 2017-
Map releaseAug 23, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8638.png

Downloads & links

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Map

FileDownload / File: emd_8638.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of Hrd1/Hrd3 dimer filtering to 4.7A and applied with -250 bfactor
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.094780415 - 0.15879112
Average (Standard dev.)-0.0000624861 (±0.008063772)
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
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.0950.159-0.000

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Supplemental data

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Additional map: map of Hrd1/Hrd3 dimer without low-pass filtering or...

Fileemd_8638_additional.map
Annotationmap of Hrd1/Hrd3 dimer without low-pass filtering or modification of amplitude
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/Hrd3 dimer

EntireName: Hrd1/Hrd3 dimer
Components
  • Complex: Hrd1/Hrd3 dimer
    • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase HRD1
  • Protein or peptide: ERAD-associated E3 ubiquitin-protein ligase component HRD3

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Supramolecule #1: Hrd1/Hrd3 dimer

SupramoleculeName: Hrd1/Hrd3 dimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's 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 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVPENRRKQL AIFVVVTYLL TFYCVYSATK TSVSFLQVTL KLNEGFNLMV LSIFILLNST LLWQLL TKL LFGELRLIEH EHIFERLPFT IINTLFMSSL FHERYFFTVA FFGLLLLYLK VFHWILKDRL EALL QSIND STTMKTLIFS RFSFNLVLLA VVDYQIITRC ...String:
MVPENRRKQL AIFVVVTYLL TFYCVYSATK TSVSFLQVTL KLNEGFNLMV LSIFILLNST LLWQLL TKL LFGELRLIEH EHIFERLPFT IINTLFMSSL FHERYFFTVA FFGLLLLYLK VFHWILKDRL EALL QSIND STTMKTLIFS RFSFNLVLLA VVDYQIITRC ISSIYTNQKS DIESTSLYLI QVMEFTMLLI DLL NLFLQT CLNFWEFYRS QQSLSNENNH IVHGDPTDEN TVESDQSQPV LNDDDDDDDD DRQFTGLEGK F MYEKAIDV FTRFLKTALH LSMLIPFRMP MMLLKDVVWD ILALYQSGTS LWKIWRNNKQ LDDTLVTV T VEQLQNSAND DNICIICMDE LIHSPNQQTW KNKNKKPKRL PCGHILHLSC LKNWMERSQT CPICRLPVFD EK

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Macromolecule #2: ERAD-associated E3 ubiquitin-protein ligase component HRD3

MacromoleculeName: ERAD-associated E3 ubiquitin-protein ligase component HRD3
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MITLLLYLCV ICNAIVLIRA DSIADPWPEA RHLLNTIAKS RDPMKEAAME PNADEFVGFY VPMDYSPRN EEKNYQSIWQ NEITDSQRHI YELLVQSSEQ FNNSEATYTL SQIHLWSQYN F PHNMTLAH KYLEKFNDLT HFTNHSAIFD LAVMYATGGC ASGNDQTVIP ...String:
MITLLLYLCV ICNAIVLIRA DSIADPWPEA RHLLNTIAKS RDPMKEAAME PNADEFVGFY VPMDYSPRN EEKNYQSIWQ NEITDSQRHI YELLVQSSEQ FNNSEATYTL SQIHLWSQYN F PHNMTLAH KYLEKFNDLT HFTNHSAIFD LAVMYATGGC ASGNDQTVIP QDSAKALLYY QR AAQLGNL KAKQVLAYKY YSGFNVPRNF HKSLVLYRDI AEQLRKSYSR DEWDIVFPYW ESY NVRISD FESGLLGKGL NSVPSSTVRK RTTRPDIGSP FIAQVNGVQM TLQIEPMGRF AFNG NDGNI NGDEDDEDAS ERRIIRIYYA ALNDYKGTYS QSRNCERAKN LLELTYKEFQ PHVDN LDPL QVFYYVRCLQ LLGHMYFTGE GSSKPNIHMA EEILTTSLEI SRRAQGPIGR ACIDLG LIN QYITNNISQA ISYYMKAMKT QANNGIVEFQ LSKLATSFPE EKIGDPFNLM ETAYLNG FI PAIYEFAVMI ESGMNSKSSV ENTAYLFKTF VDKNEAIMAP KLRTAFAALI NDRSEVAL W AYSQLAEQGY ETAQVSAAYL MYQLPYEFED PPRTTDQRKT LAISYYTRAF KQGNIDAGV VAGDIYFQMQ NYSKAMALYQ GAALKYSIQA IWNLGYMHEH GLGVNRDFHL AKRYYDQVSE HDHRFYLAS KLSVLKLHLK SWLTWITREK VNYWKPSSPL NPNEDTQHSK TSWYKQLTKI L QRMRHKED SDKAAEDSHK HRTVVQNGAN HRGDDQEEAS EILGFQMEDG GGENLYFQSG GG MDEKTTG WRGGHVVEGL AGELEQLRAR LEHHPQGQRE P

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400
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) / Average electron dose: 82.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 139754
FSC plot (resolution estimation)

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