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- EMDB-45427: HAstV1 spike in complex with neutralizing Fabs 3H4 and 3B4 -

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Basic information

Entry
Database: EMDB / ID: EMD-45427
TitleHAstV1 spike in complex with neutralizing Fabs 3H4 and 3B4
Map datasharpened map
Sample
  • Complex: Human astrovirus 1 spike in complex with Fab 3B4 and Fab 3H4
    • Complex: HAstV1 neutralizing Fab 3B4
      • Protein or peptide: HAstV1 neutralizing Fab 3B4 heavy chain
      • Protein or peptide: HAstV1 neutralizing Fab 3B4 kappa chain
    • Complex: Recombinant human astrovirus serotype 1 spike protein
      • Protein or peptide: Structural protein
    • Complex: HAstV1 neutralizing Fab 3H4
      • Protein or peptide: HAstV1 neutralizing Fab 3H4 heavy chain
      • Protein or peptide: HAstV1 neutralizing Fab 3H4 lambda chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsantibody / 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
Methodsingle particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsLanning S / DuBois RM / Balasco Serrao VH
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseDec 25, 2024-
Map releaseDec 25, 2024-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45427.png

Downloads & links

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Map

FileDownload / File: emd_45427.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.42 Å/pix.
x 686 pix.
= 284.69 Å		0.42 Å/pix.
x 686 pix.
= 284.69 Å		0.42 Å/pix.
x 686 pix.
= 284.69 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.415 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0221
Minimum - Maximum-0.27724278 - 0.36337492
Average (Standard dev.)0.000041575127 (±0.0058708275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions686686686
Spacing686686686
CellA=B=C: 284.69 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_45427_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_45427_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_45427_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Human astrovirus 1 spike in complex with Fab 3B4 and Fab 3H4

EntireName: Human astrovirus 1 spike in complex with Fab 3B4 and Fab 3H4
Components
  • Complex: Human astrovirus 1 spike in complex with Fab 3B4 and Fab 3H4
    • Complex: HAstV1 neutralizing Fab 3B4
      • Protein or peptide: HAstV1 neutralizing Fab 3B4 heavy chain
      • Protein or peptide: HAstV1 neutralizing Fab 3B4 kappa chain
    • Complex: Recombinant human astrovirus serotype 1 spike protein
      • Protein or peptide: Structural protein
    • Complex: HAstV1 neutralizing Fab 3H4
      • Protein or peptide: HAstV1 neutralizing Fab 3H4 heavy chain
      • Protein or peptide: HAstV1 neutralizing Fab 3H4 lambda chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Human astrovirus 1 spike in complex with Fab 3B4 and Fab 3H4

SupramoleculeName: Human astrovirus 1 spike in complex with Fab 3B4 and Fab 3H4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: HAstV1 neutralizing Fab 3B4

SupramoleculeName: HAstV1 neutralizing Fab 3B4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 / Details: Recombinant Fab expressed in CHO-S cells
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: Recombinant human astrovirus serotype 1 spike protein

SupramoleculeName: Recombinant human astrovirus serotype 1 spike protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Human astrovirus 1

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Supramolecule #4: HAstV1 neutralizing Fab 3H4

SupramoleculeName: HAstV1 neutralizing Fab 3H4 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: HAstV1 neutralizing Fab 3B4 heavy chain

MacromoleculeName: HAstV1 neutralizing Fab 3B4 heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c
Molecular weightTheoretical: 24.231156 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QVQLHQPGAE LVKPGASVNL SCKASGYTFT SYWMHWVKQR PGQGLEWIGE INPSSGRANY NEKFKNKATL TVDKSSITAY MHLSSLTSE DSAVYYCHWD YYAMDYWGQG TSVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS G ALTSGVHT ...String:
QVQLHQPGAE LVKPGASVNL SCKASGYTFT SYWMHWVKQR PGQGLEWIGE INPSSGRANY NEKFKNKATL TVDKSSITAY MHLSSLTSE DSAVYYCHWD YYAMDYWGQG TSVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS G ALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CASLVPR

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Macromolecule #2: HAstV1 neutralizing Fab 3B4 kappa chain

MacromoleculeName: HAstV1 neutralizing Fab 3B4 kappa chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c
Molecular weightTheoretical: 23.42085 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIVLTQSPAT LSVTPGDSVS LSCRASQSIS NNLHWYQQKS HESPRLLFKS ASQSISGIPS RFSGSGSGTD FTLSINSVET EDFGMYFCQ QTNSWPLTFG TGTKLDLKRR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIVLTQSPAT LSVTPGDSVS LSCRASQSIS NNLHWYQQKS HESPRLLFKS ASQSISGIPS RFSGSGSGTD FTLSINSVET EDFGMYFCQ QTNSWPLTFG TGTKLDLKRR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGE

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Macromolecule #3: Structural protein

MacromoleculeName: Structural protein / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Human astrovirus 1
Molecular weightTheoretical: 25.231674 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGEEYKVVLT FGSPMSPNAN NKQTWVNKPL DAPSGHYNVK IAKDVDHYLT MQGFTSIASV DWYTIDFQPS EAPAPIKGLQ VLVNISKKA DVYAVKQFVT AQTNNKHQVT SLFLVKVTTG FQVNNYLSYF YRASATGDAT TNLLVRGDTY TAGISFTQGG W YLLTNTSI ...String:
MGEEYKVVLT FGSPMSPNAN NKQTWVNKPL DAPSGHYNVK IAKDVDHYLT MQGFTSIASV DWYTIDFQPS EAPAPIKGLQ VLVNISKKA DVYAVKQFVT AQTNNKHQVT SLFLVKVTTG FQVNNYLSYF YRASATGDAT TNLLVRGDTY TAGISFTQGG W YLLTNTSI VDGAMPPGWV WNNVELKTNT AYHMDKGLVH LIMPLPESTQ MCYEMLTSIP AAAELALVPR

UniProtKB: Capsid polyprotein VP90

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Macromolecule #4: HAstV1 neutralizing Fab 3H4 heavy chain

MacromoleculeName: HAstV1 neutralizing Fab 3H4 heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c
Molecular weightTheoretical: 24.436518 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QVQLKESGPG LVAPSQSLSI SCTVSGFSLT TFGIHWIRQP PGKGLEWLGV IWAAGTTNYN STLKSRLTIT KDNSRSQVFL KMNSLQTYD TAIYYCVRED YDYFFGLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS W NSGALTSG ...String:
QVQLKESGPG LVAPSQSLSI SCTVSGFSLT TFGIHWIRQP PGKGLEWLGV IWAAGTTNYN STLKSRLTIT KDNSRSQVFL KMNSLQTYD TAIYYCVRED YDYFFGLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS W NSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCASLVPR

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Macromolecule #5: HAstV1 neutralizing Fab 3H4 lambda chain

MacromoleculeName: HAstV1 neutralizing Fab 3H4 lambda chain / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c
Molecular weightTheoretical: 23.275816 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYASWVQE KPDHLFIGLI GGTNNRAPGV PARFSGSLIG DKAALTITGA QTDDEAIYF CALWFSNHWV FGGGTKLTVL GRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE ...String:
QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYASWVQE KPDHLFIGLI GGTNNRAPGV PARFSGSLIG DKAALTITGA QTDDEAIYF CALWFSNHWV FGGGTKLTVL GRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration0.86 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
10.0 mMTris base
150.0 mMsodium chlorideNaCl
3.57 uMlauryl maltose neopentyl glycolC47H88O22

Details: TBS buffer pH 8 with 3.57 uM LMNG detergent to improve orientation bias
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7235 / Average electron dose: 32.26 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4132753
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 163237
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 3
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5ewo

chain_id: C, source_name: PDB, initial_model_type: experimental model

5ewo

chain_id: D, source_name: PDB, initial_model_type: experimental model
chain_id: A, source_name: AlphaFold, initial_model_type: in silico model
chain_id: B, source_name: AlphaFold, initial_model_type: in silico model
chain_id: E, source_name: AlphaFold, initial_model_type: in silico model
chain_id: F, source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 112
Output model

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

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