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- EMDB-21224: CryoEM map of Hrd3/Yos9 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21224
TitleCryoEM map of Hrd3/Yos9 complex
Map data
Sample
  • Complex: a complex of Hrd1/Hrd3/Yos9
    • Protein or peptide: Hrd3
    • Protein or peptide: Protein OS-9 homolog
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 / oligosaccharide binding / negative regulation of protein autoubiquitination / retrograde protein transport, ER to cytosol / ERAD pathway ...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 / oligosaccharide binding / negative regulation of protein autoubiquitination / retrograde protein transport, ER to cytosol / ERAD pathway / endoplasmic reticulum unfolded protein response / endoplasmic reticulum lumen / endoplasmic reticulum membrane / endoplasmic reticulum / identical protein binding
Similarity search - Function
Yos9, dimerzation domain / Protein OS-9-like / Yos9 dimerzation domain / Protein OS9-like domain / Glucosidase II beta subunit-like protein / Sel1 repeat / Sel1-like repeat / Sel1-like repeats. / MRH domain / MRH domain profile. ...Yos9, dimerzation domain / Protein OS-9-like / Yos9 dimerzation domain / Protein OS9-like domain / Glucosidase II beta subunit-like protein / Sel1 repeat / Sel1-like repeat / Sel1-like repeats. / MRH domain / MRH domain profile. / Mannose-6-phosphate receptor binding domain superfamily / Endoplasmic reticulum targeting sequence. / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
ERAD-associated E3 ubiquitin-protein ligase component HRD3 / Protein OS-9 homolog
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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.
History
DepositionJan 18, 2020-
Header (metadata) releaseApr 29, 2020-
Map releaseApr 29, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vk3
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21224.png

Downloads & links

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Map

FileDownload / File: emd_21224.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.028
Minimum - Maximum-0.1404696 - 0.21274345
Average (Standard dev.)0.00019457632 (±0.0045524333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 233.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z233.200233.200233.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.1400.2130.000

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Supplemental data

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Additional map: unsharpened map

Fileemd_21224_additional.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : a complex of Hrd1/Hrd3/Yos9

EntireName: a complex of Hrd1/Hrd3/Yos9
Components
  • Complex: a complex of Hrd1/Hrd3/Yos9
    • Protein or peptide: Hrd3
    • Protein or peptide: Protein OS-9 homolog

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Supramolecule #1: a complex of Hrd1/Hrd3/Yos9

SupramoleculeName: a complex of Hrd1/Hrd3/Yos9 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Hrd3

MacromoleculeName: Hrd3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 84.328023 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MITLLLYLCV ICNAIVLIRA DSIADPWPEA RHLLNTIAKS RDPMKEAAME PNADEFVGFY VPMDYSPRNE EKNYQSIWQN EITDSQRHI YELLVQSSEQ FNNSEATYTL SQIHLWSQYN FPHNMTLAHK YLEKFNDLTH FTNHSAIFDL AVMYATGGCA S GNDQTVIP ...String:
MITLLLYLCV ICNAIVLIRA DSIADPWPEA RHLLNTIAKS RDPMKEAAME PNADEFVGFY VPMDYSPRNE EKNYQSIWQN EITDSQRHI YELLVQSSEQ FNNSEATYTL SQIHLWSQYN FPHNMTLAHK YLEKFNDLTH FTNHSAIFDL AVMYATGGCA S GNDQTVIP QDSAKALLYY QRAAQLGNLK AKQVLAYKYY SGFNVPRNFH KSLVLYRDIA EQLRKSYSRD EWDIVFPYWE SY NVRISDF ESGLLGKGLN SVPSSTVRKR TTR(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) DASERRIIRI YYAAL NDYK GTYSQSRNCE RAKNLLELTY KEFQPHVDNL DPLQVFYYVR CLQLLGHMYF TGEGSSKPNI HMAEEILTTS LEISRR AQG PIGRACIDLG LINQYITNNI SQAISYYMKA MKTQANNGIV EFQLSKLATS FPEEKIGDPF NLMETAYLNG FIPAIYE FA VMIESGMNSK SSVENTAYLF KTFVDKNEAI MAPKLRTAFA ALINDRSEVA LWAYSQLAEQ GYETAQVSAA YLMYQLPY E FEDPPRTTDQ RKTLAISYYT RAFKQGNIDA GVVAGDIYFQ MQNYSKAMAL YQGAALKYSI QAIWNLGYMH EHGLGVNRD FHLAKRYYDQ VSEHDHRFYL ASKLSVLKLH LKSWLTWITR EKVNYWKPSS PLNPNEDTQH SKTSWYKQLT KILQRMRHKE DSDKAAEDS HKHRTVVQNG ANHRGDDQEE ASEILGFQME D

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Macromolecule #2: Protein OS-9 homolog

MacromoleculeName: Protein OS-9 homolog / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 61.311285 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MQAKIIYALS AISALIPLGS SLLAPIEDPI VSNKYLISYI DEDDWSDRIL QNQSVMNSGY IVNMGDDLEC FIQNASTQLN DVLEDSNEH SNSEKTALLT KTLNQGVKTI FDKLNERCIF YQAGFWIYEY CPGIEFVQFH GRVNTKTGEI VNRDESLVYR L GKPKANVE ...String:
MQAKIIYALS AISALIPLGS SLLAPIEDPI VSNKYLISYI DEDDWSDRIL QNQSVMNSGY IVNMGDDLEC FIQNASTQLN DVLEDSNEH SNSEKTALLT KTLNQGVKTI FDKLNERCIF YQAGFWIYEY CPGIEFVQFH GRVNTKTGEI VNRDESLVYR L GKPKANVE EREFELLYDD VGYYISEIIG SGDICDVTGA ERMVEIQYVC GGSNSGPSTI QWVRETKICV YEAQVTIPEL CN LELLAKN EDQKNASPIL CRMPAKSKIG SNSIDLITKY EPIFLGSGIY FLRPFNTDER DKLMVTDNAM SNWDEITETY YQK FGNAIN KMLSLRLVSL PNGHILQPGD SCVWLAEVVD MKDRFQTTLS LNILNSQRAE IFFNKTFTFN EDNGNFLSYK IGDH GESTE LGQITHSNKA DINTAEIRSD EYLINTDNEL FLRISKEIAE VKELLNEIVS PHEMEVIFEN MRNQPNNDFE LALMN KLKS SLNDDNKVEQ INNARMDDDE STSHTTRDIG EAGSQTTGNT ESEVTNVAAG VFIEHDEL

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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

Concentration6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
25.0 mMC8H18N2O4SHEPES
0.06 %C56H92O29digitonin
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
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: OTHER / Imaging mode: OTHER
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 44.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 99298

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