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- PDB-9cbn: HAstV1 spike in complex with neutralizing Fabs 3H4 and 3B4 -

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Basic information

Entry
Database: PDB / ID: 9cbn
TitleHAstV1 spike in complex with neutralizing Fabs 3H4 and 3B4
Components
  • (HAstV1 neutralizing Fab 3B4 ...) x 2
  • (HAstV1 neutralizing Fab 3H4 ...) x 2
  • Structural protein
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / antibody / virus / spike / homodimer / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homologyTurkey astrovirus capsid protein / Turkey astrovirus capsid protein / Capsid, astroviral / Astrovirus capsid protein nucleoplasmin-like domain / T=3 icosahedral viral capsid / Viral coat protein subunit / clathrin-dependent endocytosis of virus by host cell / host extracellular space / Capsid polyprotein VP90
Function and homology information
Biological speciesMus musculus (house mouse)
Human astrovirus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsLanning, S. / DuBois, R.M. / Balasco Serrao, V.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI144090 United States
CitationJournal: J Virol / Year: 2025
Title: Discovery of three novel neutralizing antibody epitopes on the human astrovirus capsid spike and mechanistic insights into virus neutralization.
Authors: Sarah Lanning / Nayeli Aguilar-Hernández / Vitor Hugo B Serrão / Tomás López / Sara M O'Rourke / Adam Lentz / Lena Ricemeyer / Rafaela Espinosa / Susana López / Carlos F Arias / Rebecca M DuBois /
Abstract: Human astroviruses (HAstVs) are a leading cause of viral childhood diarrhea that infects nearly every individual during their lifetime. Although human astroviruses are highly prevalent, no approved ...Human astroviruses (HAstVs) are a leading cause of viral childhood diarrhea that infects nearly every individual during their lifetime. Although human astroviruses are highly prevalent, no approved vaccine currently exists. Antibody responses appear to play an important role in protection from HAstV infection; however, knowledge about the neutralizing epitope landscape is lacking, as only three neutralizing antibody epitopes have previously been determined. Here, we structurally define the epitopes of three uncharacterized HAstV-neutralizing monoclonal antibodies: antibody 4B6 with X-ray crystallography to 2.67 Å, and antibodies 3H4 and 3B4 simultaneously with single-particle cryogenic-electron microscopy to 3.33 Å. We assess the epitope locations relative to conserved regions on the capsid spike and find that while antibodies 4B6 and 3B4 target the upper variable loop regions of the HAstV spike protein, antibody 3H4 targets a novel region near the base of the spike that is more conserved. Additionally, we found that all three antibodies bind with high affinity, and they compete with receptor FcRn binding to the capsid spike. These studies inform which regions of the HAstV capsid can be targeted by monoclonal antibody therapies and could aid in rational vaccine design.IMPORTANCEHuman astroviruses (HAstVs) infect nearly every child in the world, causing diarrhea, vomiting, and fever. Despite the prevalence of human astroviruses, little is known about how antibodies block virus infection. Here, we determined high-resolution structures of the astrovirus capsid protein in a complex with three virus-neutralizing antibodies. The antibodies bind distinct sites on the capsid spike domain. The antibodies block virus attachment to human cells and prevent capsid spike interaction with the human neonatal Fc receptor. These findings support the use of the human astrovirus capsid spike as an antigen in a vaccine to prevent astrovirus disease.
History
DepositionJun 19, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 25, 2024Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Dec 25, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 25, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2025Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_related_exp_data_set
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Mar 5, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update
Revision 1.1Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / Category: citation / em_admin / Data content type: EM metadata / EM metadata / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HAstV1 neutralizing Fab 3B4 heavy chain
B: HAstV1 neutralizing Fab 3B4 kappa chain
C: Structural protein
D: Structural protein
E: HAstV1 neutralizing Fab 3H4 heavy chain
F: HAstV1 neutralizing Fab 3H4 lambda chain
G: HAstV1 neutralizing Fab 3H4 heavy chain
H: HAstV1 neutralizing Fab 3H4 lambda chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,93411
Polymers193,5408
Non-polymers1,3943
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 2 molecules CD

#3: Protein Structural protein / spike


Mass: 25231.674 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human astrovirus 1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q82452

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Antibody , 4 types, 6 molecules ABEGFH

#1: Antibody HAstV1 neutralizing Fab 3B4 heavy chain


Mass: 24231.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: BALB/c / Cell line (production host): CHO-S / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody HAstV1 neutralizing Fab 3B4 kappa chain


Mass: 23420.850 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: BALB/c / Cell line (production host): CHO-S / Production host: Cricetulus griseus (Chinese hamster)
#4: Antibody HAstV1 neutralizing Fab 3H4 heavy chain


Mass: 24436.518 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: BALB/c / Cell line (production host): CHO-S / Production host: Cricetulus griseus (Chinese hamster)
#5: Antibody HAstV1 neutralizing Fab 3H4 lambda chain


Mass: 23275.816 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: BALB/c / Cell line (production host): CHO-S / Production host: Cricetulus griseus (Chinese hamster)

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Sugars , 2 types, 3 molecules

#6: 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.: 2
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
#7: 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.: 1 / 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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Human astrovirus 1 spike in complex with Fab 3B4 and Fab 3H4COMPLEX#1-#50MULTIPLE SOURCES
2HAstV1 neutralizing Fab 3B4COMPLEX#1-#21RECOMBINANTRecombinant Fab expressed in CHO-S cells
3Recombinant human astrovirus serotype 1 spike proteinCOMPLEX#31RECOMBINANT
4HAstV1 neutralizing Fab 3H4COMPLEX#4-#51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Human astrovirus 112456
34Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Cricetulus griseus (Chinese hamster)10029CHO-S
23Escherichia coli (E. coli)562
34Cricetulus griseus (Chinese hamster)10029CHO-S
Buffer solutionpH: 8
Details: TBS buffer pH 8 with 3.57 uM LMNG detergent to improve orientation bias
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris base1
2150 mMsodium chlorideNaCl1
33.57 uMlauryl maltose neopentyl glycolC47H88O221
SpecimenConc.: 0.86 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: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 32.26 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7235

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4132753
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163237 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 112 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDSource nameType
15EWO

5ewo

C5EWOC1PDBexperimental model
25EWO

5ewo

D5EWOD1PDBexperimental model
3AAlphaFoldin silico model
4BAlphaFoldin silico model
5EAlphaFoldin silico model
6FAlphaFoldin silico model

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