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- PDB-7v0n: Cryo-EM structure of SINV/EEEV in complex with Fab fragment of a ... -

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Basic information

Entry
Database: PDB / ID: 7v0n
TitleCryo-EM structure of SINV/EEEV in complex with Fab fragment of a moderately/weakly neutralizing human antibody IgG-21
Components
  • IgG 21 Fab heavy chain
  • IgG 21 Fab light chain
  • Spike glycoprotein E1
  • Spike glycoprotein E2
KeywordsVIRUS/IMMUNE SYSTEM / cryo-EM / single particle / virus neutralization / inter virion crosslink / aggregation / VIRUS-IMMUNE SYSTEM complex
Function / homology
Function and homology information


togavirin / T=4 icosahedral viral capsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / host cell cytoplasm / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / host cell nucleus / virion attachment to host cell ...togavirin / T=4 icosahedral viral capsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / host cell cytoplasm / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / host cell nucleus / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / RNA binding / membrane
Similarity search - Function
Alphavirus E2 glycoprotein, domain B / Peptidase S3, togavirin / Alphavirus E2 glycoprotein / Alphavirus E3 spike glycoprotein / Alphavirus E1 glycoprotein / Alphavirus E2 glycoprotein, domain A / Alphavirus E2 glycoprotein, domain C / Alphavirus E2 glycoprotein / Alphavirus core protein / Alphavirus E3 glycoprotein ...Alphavirus E2 glycoprotein, domain B / Peptidase S3, togavirin / Alphavirus E2 glycoprotein / Alphavirus E3 spike glycoprotein / Alphavirus E1 glycoprotein / Alphavirus E2 glycoprotein, domain A / Alphavirus E2 glycoprotein, domain C / Alphavirus E2 glycoprotein / Alphavirus core protein / Alphavirus E3 glycoprotein / Alphavirus E1 glycoprotein / Alphavirus core protein (CP) domain profile. / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Flaviviral glycoprotein E, dimerisation domain / Immunoglobulin E-set / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Structural polyprotein
Similarity search - Component
Biological speciesEastern equine encephalitis virus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsBandyopadhyay, A. / Klose, T. / Kuhn, R.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI142790 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI095366 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies.
Authors: Lauren E Williamson / Abhishek Bandyopadhyay / Kevin Bailey / Devika Sirohi / Thomas Klose / Justin G Julander / Richard J Kuhn / James E Crowe /
Abstract: Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory ...Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.
History
DepositionMay 10, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 6, 2023Group: Data collection / Source and taxonomy / Category: chem_comp_atom / chem_comp_bond / entity_src_gen
Item: _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.2Feb 21, 2024Group: Refinement description / Structure summary / Category: em_3d_fitting_list / entity / pdbx_entry_details
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _entity.pdbx_fragment

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein E1
B: Spike glycoprotein E1
C: Spike glycoprotein E1
D: Spike glycoprotein E1
E: IgG 21 Fab heavy chain
F: IgG 21 Fab light chain
G: IgG 21 Fab heavy chain
H: IgG 21 Fab light chain
I: IgG 21 Fab heavy chain
J: IgG 21 Fab light chain
K: IgG 21 Fab heavy chain
L: IgG 21 Fab light chain
a: Spike glycoprotein E2
b: Spike glycoprotein E2
c: Spike glycoprotein E2
d: Spike glycoprotein E2


Theoretical massNumber of molelcules
Total (without water)481,47916
Polymers481,47916
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein
Spike glycoprotein E1 / E1 envelope glycoprotein


Mass: 43628.023 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eastern equine encephalitis virus / Strain: Florida 93-939 / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: Q4QXJ7
#2: Antibody
IgG 21 Fab heavy chain


Mass: 20017.594 Da / Num. of mol.: 4
Fragment: Heavy chain variable domain: QVQLVESGGGVVQPGRSLRLSCAASGFTFRTYALHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFVQMNSLRPEDTAVYYCTRVVNFGVAFIRDGVYGHYYYGMDVWGQGTTVTVSSAS
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody
IgG 21 Fab light chain


Mass: 18400.619 Da / Num. of mol.: 4
Fragment: Light chain variable domain: QSVLTQPPSVSAAPGQKVSISCSGSTSNIGNNYVSWYRQFPGTAPKFLIYDNDKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSVWVFGGGTKLTVLGQPKAAPS
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Protein
Spike glycoprotein E2 / E2 envelope glycoprotein


Mass: 38323.488 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eastern equine encephalitis virus / Strain: Florida 93-939 / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: Q4QXJ7
Sequence detailsIgG EEEV-21: Heavy chain variable domain: ...IgG EEEV-21: Heavy chain variable domain: QVQLVESGGGVVQPGRSLRLSCAASGFTFRTYALHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFVQMNSLRPEDTAVYYCTRVVNFGVAFIRDGVYGHYYYGMDVWGQGTTVTVSSAS Light chain variable domain: QSVLTQPPSVSAAPGQKVSISCSGSTSNIGNNYVSWYRQFPGTAPKFLIYDNDKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSVWVFGGGTKLTVLGQPKAAPS

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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: Homo sapiensHuman / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Eastern equine encephalitis virus11021FL93-939
31Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Mesocricetus auratus (golden hamster)10036
31Homo sapiens (human)9606
Details of virusEmpty: NO / Enveloped: YES / Isolate: OTHER / Type: VIRION
Natural hostOrganism: Culiseta melanura
Virus shellName: EEEVEastern equine encephalitis / Diameter: 850 nm / Triangulation number (T number): 4
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationCryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 38.33 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.3/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Linux / Type: package
EM software
IDNameVersionCategory
2Leginon3.2image acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
8Situsmodel fitting
10jsprinitial Euler assignment
11jsprfinal Euler assignment
12jsprclassification
13jspr3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10486
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6911 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6MX4

6mx4


Accession code: 6MX4 / Source name: PDB / Type: experimental model

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