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- PDB-8fsj: Cryo-EM structure of engineered hepatitis C virus E1E2 ectodomain... -

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Basic information

Entry
Database: PDB / ID: 8fsj
TitleCryo-EM structure of engineered hepatitis C virus E1E2 ectodomain in complex with antibodies AR4A, HEPC74, and IGH520
Components
  • AR4A heavy chain
  • AR4A light chain
  • HCV E1 ectodomain, SYNZIP1 scaffold fusion
  • HCV E2 ectodomain, SYNZIP2 scaffold fusion
  • HEPC74 heavy chain
  • HEPC74 light chain
KeywordsVIRAL PROTEIN / HCV / E1E2
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsMetcalf, M.C. / Ofek, G.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 19, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: HCV E2 ectodomain, SYNZIP2 scaffold fusion
H: HEPC74 heavy chain
L: HEPC74 light chain
A: HCV E1 ectodomain, SYNZIP1 scaffold fusion
B: AR4A light chain
C: AR4A heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,34320
Polymers174,6486
Non-polymers5,69514
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules EAC

#1: Protein HCV E2 ectodomain, SYNZIP2 scaffold fusion


Mass: 42975.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hepacivirus C, (gene. exp.) synthetic construct (others)
Strain: 1b09 / Plasmid: pCMV / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Tissue (production host): kidney / References: UniProt: A0A2P0NE15
#4: Protein HCV E1 ectodomain, SYNZIP1 scaffold fusion


Mass: 22951.379 Da / Num. of mol.: 1 / Fragment: residues 22-179
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hepacivirus C, (gene. exp.) synthetic construct (others)
Strain: 1b09 / Plasmid: pCMV / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Tissue (production host): kidney / References: UniProt: A0A2P0NE34
#6: Protein AR4A heavy chain


Mass: 31862.697 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pCMV / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Tissue (production host): kidney

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Antibody , 3 types, 3 molecules HLB

#2: Antibody HEPC74 heavy chain


Mass: 27644.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pCMV / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Tissue (production host): kidney
#3: Antibody HEPC74 light chain


Mass: 23470.111 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pCMV / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Tissue (production host): kidney
#5: Antibody AR4A light chain


Mass: 25743.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pCMV / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Tissue (production host): kidney

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Sugars , 3 types, 14 molecules

#7: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#8: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#9: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: HCV E1E2 ectodomain in complex with AR4A, HEPC74, and IGH520
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.250 MDa / Experimental value: NO
Source (natural)Organism: Hepacivirus C
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 47 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCv3.3.2particle selection
4cryoSPARCv3.3.2CTF correction
13cryoSPARCv3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2620845
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143576 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310096
ELECTRON MICROSCOPYf_angle_d0.55913779
ELECTRON MICROSCOPYf_dihedral_angle_d4.8661543
ELECTRON MICROSCOPYf_chiral_restr0.0441634
ELECTRON MICROSCOPYf_plane_restr0.0051732

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