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- PDB-9ck8: Lineage IV Lassa virus glycoprotein (Josiah) in complex with poly... -

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Basic information

Entry
Database: PDB / ID: 9ck8
TitleLineage IV Lassa virus glycoprotein (Josiah) in complex with polyclonal antibody (GPC-A epitope) from rabbit 189
Components
  • (Glycoprotein ...) x 2
  • (Rabbit polyclonal Fv ...) x 2
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / Lassa / polyclonal antibody / LL-1 / glycoprotein / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


host cell Golgi membrane / receptor-mediated endocytosis of virus by host cell / host cell endoplasmic reticulum membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / membrane / metal ion binding
Similarity search - Function
Arenavirus glycoprotein, zinc binding domain / Arenavirus glycoprotein / Arenavirus glycoprotein
Similarity search - Domain/homology
Pre-glycoprotein polyprotein GP complex
Similarity search - Component
Biological speciesLassa virus Josiah
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsBrouwer, P.J.M. / Perrett, H.R. / Ward, A.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI171438 United States
CitationJournal: Cell Rep / Year: 2024
Title: Defining bottlenecks and opportunities for Lassa virus neutralization by structural profiling of vaccine-induced polyclonal antibody responses.
Authors: Philip J M Brouwer / Hailee R Perrett / Tim Beaumont / Haye Nijhuis / Sabine Kruijer / Judith A Burger / Ilja Bontjer / Wen-Hsin Lee / James A Ferguson / Martin Schauflinger / Helena Müller- ...Authors: Philip J M Brouwer / Hailee R Perrett / Tim Beaumont / Haye Nijhuis / Sabine Kruijer / Judith A Burger / Ilja Bontjer / Wen-Hsin Lee / James A Ferguson / Martin Schauflinger / Helena Müller-Kräuter / Rogier W Sanders / Thomas Strecker / Marit J van Gils / Andrew B Ward /
Abstract: Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus ...Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccine candidates have generally been unsuccessful at doing so, and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron microscopy-based epitope mapping workflow that enables high-resolution structural characterization of polyclonal antibodies to the GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization that involve epitopes of the GPC-A competition cluster. Furthermore, by identifying undescribed immunogenic off-target epitopes, we expose the challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.
History
DepositionJul 8, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Rabbit polyclonal Fv heavy chain
L: Rabbit polyclonal Fv light chain
C: Glycoprotein GP1
A: Glycoprotein GP1
B: Glycoprotein GP1
a: Glycoprotein G2
b: Glycoprotein G2
c: Glycoprotein G2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)257,52041
Polymers245,3968
Non-polymers12,12433
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Glycoprotein ... , 2 types, 6 molecules CABabc

#3: Protein Glycoprotein GP1


Mass: 29010.352 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lassa virus Josiah / Gene: GPC / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P08669
#4: Protein Glycoprotein G2 / GP2


Mass: 46421.793 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lassa virus Josiah / Gene: GPC, GP-C / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P08669

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Antibody , 2 types, 2 molecules HL

#1: Antibody Rabbit polyclonal Fv heavy chain


Mass: 10060.393 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit)
#2: Antibody Rabbit polyclonal Fv light chain


Mass: 9039.134 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit)

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Sugars , 5 types, 33 molecules

#5: 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.: 11
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
#6: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 570.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-1-2/a4-b1_a6-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#7: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#8: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-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.: 17 / Source method: obtained synthetically / Formula: C8H15NO6
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 interestN
Has protein modificationY

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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: Lineage IV Lassa virus glycoprotein (Josiah) in complex with rabbit polyclonal antibody (GPC-A-2 epitope)
Type: COMPLEX / Entity ID: #1-#4 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Oryctolagus cuniculus (rabbit)9986
31Oryctolagus cuniculus (rabbit)9986
Source (recombinant)Organism: Homo (humans)
Buffer solutionpH: 7.4
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 41.21 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM software
IDNameCategoryDetails
4cryoSPARCCTF correctionPatchCTF
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86915 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00510135
ELECTRON MICROSCOPYf_angle_d0.83813762
ELECTRON MICROSCOPYf_dihedral_angle_d13.034313
ELECTRON MICROSCOPYf_chiral_restr0.0571748
ELECTRON MICROSCOPYf_plane_restr0.0141676

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