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- PDB-9cu7: Structure of 16.ND.92 Fab in complex with A/Solomon Islands/3/200... -

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Basic information

Entry
Database: PDB / ID: 9cu7
TitleStructure of 16.ND.92 Fab in complex with A/Solomon Islands/3/2006(H1N1) influenza virus Hemagglutinin
Components
  • Hemagglutinin HA1
  • Hemagglutinin HA2
  • Variable Heavy Chain of 16.ND.92 Fab
  • Variable Light Chain of 16.ND.92 Fab
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / Antibody / Influenza / Hemagglutinin / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / membrane
Similarity search - Function
Haemagglutinin, influenzavirus A / Haemagglutinin, HA1 chain, alpha/beta domain superfamily / Haemagglutinin / Haemagglutinin, influenzavirus A/B / Viral capsid/haemagglutinin protein
Similarity search - Domain/homology
Hemagglutinin / Hemagglutinin
Similarity search - Component
Biological speciesHomo sapiens (human)
Influenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsOuyang, W.O. / Pholcharee, T. / Wu, N.C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Complementary and Integrative Health (NIH/NCCIH)DP2 AT011966 United States
CitationJournal: Sci Transl Med / Year: 2025
Title: High-throughput synthesis and specificity characterization of natively paired influenza hemagglutinin antibodies with oPool display.
Authors: Wenhao O Ouyang / Huibin Lv / Wenkan Liu / Ruipeng Lei / Zongjun Mou / Tossapol Pholcharee / Logan Talmage / Meixuan Tong / Wei Ji / Yiquan Wang / Katrine E Dailey / Akshita B Gopal / Danbi ...Authors: Wenhao O Ouyang / Huibin Lv / Wenkan Liu / Ruipeng Lei / Zongjun Mou / Tossapol Pholcharee / Logan Talmage / Meixuan Tong / Wei Ji / Yiquan Wang / Katrine E Dailey / Akshita B Gopal / Danbi Choi / Madison R Ardagh / Lucia A Rodriguez / Jenna J Guthmiller / Xinghong Dai / Nicholas C Wu /
Abstract: Antibody discovery is crucial for developing therapeutics and vaccines and for understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a ...Antibody discovery is crucial for developing therapeutics and vaccines and for understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we present oPool display, a high-throughput cell-free platform that combined oligo pool synthesis and mRNA display to rapidly construct and characterize hundreds to thousands of natively paired antibodies in parallel. As a proof of concept, we applied oPool display to probe the binding specificity of more than 300 uncommon influenza hemagglutinin-specific antibodies against nine hemagglutinin variants through 16 screens. More than 5000 binding tests were performed in 3 to 5 days of hands-on time with further scaling potential. Follow-up structural and functional analysis of two antibodies revealed the versatility of the human immunoglobulin gene segment D3-3 () in recognizing the hemagglutinin stem. Overall, this study established an experimental platform that not only accelerates antibody characterization but also enables unbiased discovery of recurring molecular signatures among antibodies with the same specificity.
History
DepositionJul 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 30, 2025Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Nov 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Variable Heavy Chain of 16.ND.92 Fab
I: Variable Heavy Chain of 16.ND.92 Fab
J: Variable Heavy Chain of 16.ND.92 Fab
L: Variable Light Chain of 16.ND.92 Fab
M: Variable Light Chain of 16.ND.92 Fab
N: Variable Light Chain of 16.ND.92 Fab
A: Hemagglutinin HA1
C: Hemagglutinin HA1
E: Hemagglutinin HA1
B: Hemagglutinin HA2
D: Hemagglutinin HA2
F: Hemagglutinin HA2


Theoretical massNumber of molelcules
Total (without water)242,17012
Polymers242,17012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, size exclusion chromatography indicated the correct assembly
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody Variable Heavy Chain of 16.ND.92 Fab


Mass: 13659.272 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi 293F / Production host: Homo sapiens (human)
#2: Antibody Variable Light Chain of 16.ND.92 Fab


Mass: 11800.195 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi 293F / Production host: Homo sapiens (human)
#3: Protein Hemagglutinin HA1


Mass: 35851.199 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Solomon Islands/3/2006(H1N1))
Gene: HA / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0G2RTI0
#4: Protein Hemagglutinin HA2


Mass: 19412.537 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Solomon Islands/3/2006(H1N1))
Gene: HA / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: C8CQF2
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: 16.ND.92 Fab in complex with A/Solomon Islands/3/2006 (H1N1) Hemagglutinin
Type: COMPLEX
Details: Both Hemagglutinin and Fab were recombinantly expressed and purified. The Fab was then mixed with Hemagglutinin at 3.5:1 ratio. The complex were then isolated using size exclusion chromatography.
Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F
Buffer solutionpH: 7.5 / Details: 20mM Tris-HCl, 150mM NaCl
SpecimenConc.: 3 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: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K
Details: Blot force: 0, Blot time: 3 s, Humidity 90%, and 4 C.

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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: 5000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 57.35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
4cryoSPARC4.5.3CTF correction
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
13cryoSPARC4.5.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152449 / Symmetry type: POINT

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