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- EMDB-29419: Cryo-EM structure of engineered hepatitis C virus E1E2 ectodomain... -

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Entry
Database: EMDB / ID: EMD-29419
TitleCryo-EM structure of engineered hepatitis C virus E1E2 ectodomain in complex with antibodies AR4A, HEPC74, and IGH520
Map dataPrimary map
Sample
  • Complex: HCV E1E2 ectodomain in complex with AR4A, HEPC74, and IGH520
    • Protein or peptide: HCV E2 ectodomain, SYNZIP2 scaffold fusion
    • Protein or peptide: HEPC74 heavy chain
    • Protein or peptide: HEPC74 light chain
    • Protein or peptide: HCV E1 ectodomain, SYNZIP1 scaffold fusion
    • Protein or peptide: AR4A light chain
    • Protein or peptide: AR4A heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsHCV / E1E2 / VIRAL PROTEIN
Function / homology
Function and homology information


host cell mitochondrial membrane / host cell lipid droplet / host cell mitochondrion / lipid droplet / viral nucleocapsid / clathrin-dependent endocytosis of virus by host cell / host cell endoplasmic reticulum membrane / ribonucleoprotein complex / virus-mediated perturbation of host defense response / viral envelope ...host cell mitochondrial membrane / host cell lipid droplet / host cell mitochondrion / lipid droplet / viral nucleocapsid / clathrin-dependent endocytosis of virus by host cell / host cell endoplasmic reticulum membrane / ribonucleoprotein complex / virus-mediated perturbation of host defense response / viral envelope / host cell nucleus / virion membrane / structural molecule activity / membrane / cytoplasm
Similarity search - Function
Hepatitis C virus, Core protein, C-terminal / Hepatitis C virus core protein / Hepatitis C virus, Non-structural protein E2/NS1 / Hepatitis C virus non-structural protein E2/NS1 / Hepatitis C virus, Envelope glycoprotein E1 / Hepatitis C virus envelope glycoprotein E1
Similarity search - Domain/homology
Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesHepacivirus C / synthetic construct (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsMetcalf MC / Ofek G
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI168048-01 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structure of engineered hepatitis C virus E1E2 ectodomain in complex with neutralizing antibodies.
Authors: Matthew C Metcalf / Benjamin M Janus / Rui Yin / Ruixue Wang / Johnathan D Guest / Edwin Pozharski / Mansun Law / Roy A Mariuzza / Eric A Toth / Brian G Pierce / Thomas R Fuerst / Gilad Ofek /
Abstract: Hepatitis C virus (HCV) is a major global health burden as the leading causative agent of chronic liver disease and hepatocellular carcinoma. While the main antigenic target for HCV-neutralizing ...Hepatitis C virus (HCV) is a major global health burden as the leading causative agent of chronic liver disease and hepatocellular carcinoma. While the main antigenic target for HCV-neutralizing antibodies is the membrane-associated E1E2 surface glycoprotein, the development of effective vaccines has been hindered by complications in the biochemical preparation of soluble E1E2 ectodomains. Here, we present a cryo-EM structure of an engineered, secreted E1E2 ectodomain of genotype 1b in complex with neutralizing antibodies AR4A, HEPC74, and IGH520. Structural characterization of the E1 subunit and C-terminal regions of E2 reveal an overall architecture of E1E2 that concurs with that observed for non-engineered full-length E1E2. Analysis of the AR4A epitope within a region of E2 that bridges between the E2 core and E1 defines the structural basis for its broad neutralization. Our study presents the structure of an E1E2 complex liberated from membrane via a designed scaffold, one that maintains all essential structural features of native E1E2. The study advances the understanding of the E1E2 heterodimer structure, crucial for the rational design of secreted E1E2 antigens in vaccine development.
History
DepositionJan 10, 2023-
Header (metadata) releaseJul 19, 2023-
Map releaseJul 19, 2023-
UpdateJul 19, 2023-
Current statusJul 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29419.png

Downloads & links

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Map

FileDownload / File: emd_29419.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map
Voxel sizeX=Y=Z: 0.889 Å
Density
Contour LevelBy AUTHOR: 0.0825
Minimum - Maximum-1.0854912 - 1.576691
Average (Standard dev.)-0.00015701722 (±0.020844437)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 320.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Half map A for local refinement map

Fileemd_29419_additional_1.map
AnnotationHalf map A for local refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened local refinement map

Fileemd_29419_additional_2.map
AnnotationSharpened local refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Half map B for local refinement map

Fileemd_29419_additional_3.map
AnnotationHalf map B for local refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Non-sharpened local refinement map

Fileemd_29419_additional_4.map
AnnotationNon-sharpened local refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Merged primary and local refinement map

Fileemd_29419_additional_5.map
AnnotationMerged primary and local refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Non-sharpened primary map

Fileemd_29419_additional_6.map
AnnotationNon-sharpened primary map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B for primary map

Fileemd_29419_half_map_1.map
AnnotationHalf map B for primary map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A for primary map

Fileemd_29419_half_map_2.map
AnnotationHalf map A for primary 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 : HCV E1E2 ectodomain in complex with AR4A, HEPC74, and IGH520

EntireName: HCV E1E2 ectodomain in complex with AR4A, HEPC74, and IGH520
Components
  • Complex: HCV E1E2 ectodomain in complex with AR4A, HEPC74, and IGH520
    • Protein or peptide: HCV E2 ectodomain, SYNZIP2 scaffold fusion
    • Protein or peptide: HEPC74 heavy chain
    • Protein or peptide: HEPC74 light chain
    • Protein or peptide: HCV E1 ectodomain, SYNZIP1 scaffold fusion
    • Protein or peptide: AR4A light chain
    • Protein or peptide: AR4A heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: HCV E1E2 ectodomain in complex with AR4A, HEPC74, and IGH520

SupramoleculeName: HCV E1E2 ectodomain in complex with AR4A, HEPC74, and IGH520
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Hepacivirus C
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: HCV E2 ectodomain, SYNZIP2 scaffold fusion

MacromoleculeName: HCV E2 ectodomain, SYNZIP2 scaffold fusion / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 42.975371 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: STHVTGGTAS HTTRHFASLF SSGASQRVQL INTNGSWHIN RTALNCNDSL HTGFLAALFY THKFNASGCP ERMAHCRPID EFAQGWGPI TYAEGHGSDQ RPYCWHYAPR QCGTIPASQV CGPVYCFTPS PVVVGTTDRF GAPTYTWGEN ETDVLILNNT R PPQGNWFG ...String:
STHVTGGTAS HTTRHFASLF SSGASQRVQL INTNGSWHIN RTALNCNDSL HTGFLAALFY THKFNASGCP ERMAHCRPID EFAQGWGPI TYAEGHGSDQ RPYCWHYAPR QCGTIPASQV CGPVYCFTPS PVVVGTTDRF GAPTYTWGEN ETDVLILNNT R PPQGNWFG CTWMNSTGFT KTCGGPPCNI GGVGNNTLTC PTDCFRKHPE ATYTKCGSGP WLTPRCLVDY PYRLWHYPCT VN FTIFKVR MYVGGVEHRL NAACNWTRGE RCDLQDRDRS ELSPLLLSTT EWQILPCSFT TLPALSTGLI HLHQNIVDVQ YLY GIGSAV VSFAIPGGLV AQLENEVASL ENENETLKKK NLHKKDLIAY LEKEIANLRK KIEHHHHHH

UniProtKB: Genome polyprotein

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Macromolecule #2: HEPC74 heavy chain

MacromoleculeName: HEPC74 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.644807 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGAE VKKPGSSVKV SCTTSGGTYI NYAISWVRQA PGQGLEWVGG MSPISNTPKY AQKFQGRVTI TADESTSTTY MELSSLRPE DTAVYYCARD LLKYCGGGNC HSLLVDPWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
QVQLVQSGAE VKKPGSSVKV SCTTSGGTYI NYAISWVRQA PGQGLEWVGG MSPISNTPKY AQKFQGRVTI TADESTSTTY MELSSLRPE DTAVYYCARD LLKYCGGGNC HSLLVDPWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTAGWSH PQ FEKGGGS GGGSGGSSAW SHPQFEK

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Macromolecule #3: HEPC74 light chain

MacromoleculeName: HEPC74 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.470111 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIVMTQSPST LSASVGDRVT ISCRASQSIS SWLAWYQQKP GRAPKLLIYK ASSLETGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQ HYNTYLFTFG PGTKVDLKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIVMTQSPST LSASVGDRVT ISCRASQSIS SWLAWYQQKP GRAPKLLIYK ASSLETGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQ HYNTYLFTFG PGTKVDLKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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Macromolecule #4: HCV E1 ectodomain, SYNZIP1 scaffold fusion

MacromoleculeName: HCV E1 ectodomain, SYNZIP1 scaffold fusion / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 22.951379 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: YEVRNASGLY HVTNDCSNAS IVYETTDMIM HTPGCVPCVR EDNSSRCWVA LTPTLAARNA SVPTVAIRRH VDLLVGAAAF CSAMYVGDL CGSVFLVSQL FTFSPRRHET VQDCNCSIYP GHVSGHRMAW DMMMNWSPTA ALVVSQLLRI PQAVVDMVAP G GRNAYLRK ...String:
YEVRNASGLY HVTNDCSNAS IVYETTDMIM HTPGCVPCVR EDNSSRCWVA LTPTLAARNA SVPTVAIRRH VDLLVGAAAF CSAMYVGDL CGSVFLVSQL FTFSPRRHET VQDCNCSIYP GHVSGHRMAW DMMMNWSPTA ALVVSQLLRI PQAVVDMVAP G GRNAYLRK KIARLKKDNL QLERDEQNLE KIIANLRDEI ARLENEVA

UniProtKB: Genome polyprotein

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Macromolecule #5: AR4A light chain

MacromoleculeName: AR4A light chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.743781 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGVPTQVLGL LLLWLTDARC EIELTLTQSP GTLSLSPGER ATLSCRASQS VSNNYLAWYQ QKPGQAPRLL IYGASSRATG IPDRFSGSG SGTGFTLIIS RLEPEDFAVY YCQQYGSSSI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL N NFYREAKV ...String:
MGVPTQVLGL LLLWLTDARC EIELTLTQSP GTLSLSPGER ATLSCRASQS VSNNYLAWYQ QKPGQAPRLL IYGASSRATG IPDRFSGSG SGTGFTLIIS RLEPEDFAVY YCQQYGSSSI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL N NFYREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

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Macromolecule #6: AR4A heavy chain

MacromoleculeName: AR4A heavy chain / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31.862697 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MYRMQLLSCI ALSLALVTNS EVQLLEQSGP EVKKPGDSLR ISCKMSGDSL VTTWIGWVRQ KPGQGLEWMG IINPGDSSTN IYPGDSATR YGPSFQGQVT ISIDKSTSTA YLQWNNVKAS DTGIYYCARH VPVPISGTFL WREREMHDFG YFDDWGQGTL V IVSSASTK ...String:
MYRMQLLSCI ALSLALVTNS EVQLLEQSGP EVKKPGDSLR ISCKMSGDSL VTTWIGWVRQ KPGQGLEWMG IINPGDSSTN IYPGDSATR YGPSFQGQVT ISIDKSTSTA YLQWNNVKAS DTGIYYCARH VPVPISGTFL WREREMHDFG YFDDWGQGTL V IVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SL GTQTYIC NVNHKPSNTK VDKKVEPKSC AGWSHPQFEK GGGSGGGSGG SSAWSHPQFE K

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Macromolecule #9: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 9 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration1 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.0 e/Å2

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Image processing

Particle selectionNumber selected: 2620845
Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.2) / Number images used: 143576

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