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- PDB-9cn2: Human astrovirus 2 spike bound to neutralizing scFv 4B6 -

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Basic information

Entry
Database: PDB / ID: 9cn2
TitleHuman astrovirus 2 spike bound to neutralizing scFv 4B6
Components
  • Neutralizing scFv 4B6
  • spike protein
KeywordsVIRAL PROTEIN / Antibody / spike / homodimer / neutralization
Biological speciesHuman astrovirus 2
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.67 Å
AuthorsLanning, S. / DuBois, R.M.
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
DepositionJul 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 25, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 9, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: spike protein
B: Neutralizing scFv 4B6


Theoretical massNumber of molelcules
Total (without water)52,5672
Polymers52,5672
Non-polymers00
Water543
1
A: spike protein
B: Neutralizing scFv 4B6

A: spike protein
B: Neutralizing scFv 4B6


Theoretical massNumber of molelcules
Total (without water)105,1344
Polymers105,1344
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation18_454-x-1/4,z+1/4,y-1/41
Unit cell
Length a, b, c (Å)160.348, 160.348, 160.348
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number212
Space group name H-MP4332

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Components

#1: Protein spike protein


Mass: 26047.693 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human astrovirus 2 / Production host: Escherichia coli BL21 (bacteria)
#2: Antibody Neutralizing scFv 4B6


Mass: 26519.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Strain: BALB/c / Cell (production host): CHO-S / Production host: Cricetulus griseus (Chinese hamster)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.27 Å3/Da / Density % sol: 62.36 % / Description: 4 sided diamond shape
Crystal growTemperature: 295.15 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 0.1 M Tris-HCl pH 8.5, and 0.74 M sodium citrate pH 5.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 1.0332 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jun 24, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 2.67→160.64 Å / Num. obs: 20653 / % possible obs: 100 % / Redundancy: 74.5 % / CC1/2: 0.999 / Rmerge(I) obs: 0.191 / Net I/σ(I): 23.4
Reflection shellResolution: 2.67→2.72 Å / Rmerge(I) obs: 3.291 / Num. unique obs: 1020 / CC1/2: 0.755 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.20.1-4487)refinement
DIALSdata reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.67→42.85 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 27.95 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2593 1997 9.7 %
Rwork0.2122 --
obs0.2168 20583 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.67→42.85 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3493 0 0 3 3496
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.009
X-RAY DIFFRACTIONf_angle_d1.069
X-RAY DIFFRACTIONf_dihedral_angle_d6.662479
X-RAY DIFFRACTIONf_chiral_restr0.059539
X-RAY DIFFRACTIONf_plane_restr0.008617
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.67-2.740.33181390.30361294X-RAY DIFFRACTION100
2.74-2.810.35861400.27361299X-RAY DIFFRACTION100
2.81-2.890.31361380.28671287X-RAY DIFFRACTION100
2.89-2.990.34231420.29081319X-RAY DIFFRACTION100
2.99-3.090.41461390.30631289X-RAY DIFFRACTION100
3.09-3.220.38651390.30821298X-RAY DIFFRACTION100
3.22-3.360.29241390.26311315X-RAY DIFFRACTION100
3.36-3.540.30251420.23321308X-RAY DIFFRACTION100
3.54-3.760.24511400.2361315X-RAY DIFFRACTION100
3.76-4.050.24831440.21891334X-RAY DIFFRACTION100
4.06-4.460.22791430.17011328X-RAY DIFFRACTION100
4.46-5.10.20741440.14861342X-RAY DIFFRACTION100
5.11-6.410.21621490.18391379X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: -21.3371 Å / Origin y: 20.8602 Å / Origin z: -10.2538 Å
111213212223313233
T0.6335 Å20.0377 Å20.0239 Å2-0.3645 Å2-0.0993 Å2--0.3822 Å2
L2.1328 °2-0.3468 °21.0708 °2-0.3083 °2-0.8495 °2--1.4755 °2
S-0.0474 Å °-0.2887 Å °0.0174 Å °0.1856 Å °0.0427 Å °-0.0184 Å °-0.2248 Å °-0.145 Å °-0.0043 Å °
Refinement TLS groupSelection details: all

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