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- EMDB-17724: CryoEM reconstruction of hemagglutinin HK68 of Influenza A virus ... -

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Basic information

Entry
Database: EMDB / ID: EMD-17724
TitleCryoEM reconstruction of hemagglutinin HK68 of Influenza A virus bound to an Affimer reagent
Map data
Sample
  • Complex: CryoEM reconstruction of hemagglutinin HK68 of Influenza A virus bound to an Affimer reagent
    • Protein or peptide: Hemagglutinin HA2 chain
    • Protein or peptide: Affimer molecule (A31)
  • Protein or peptide: Hemagglutinin HA1 chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsComplex / Inhibitor / CryoEM / Antiviral / ANTIVIRAL PROTEIN
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 speciesInfluenza A virus / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDebski-Antoniak O / Flynn A / Klebl DP / Tiede C / Muench S / Tomlinson D / Fontana J
Funding support1 items
OrganizationGrant numberCountry
Wellcome TrustSpringboard Award
CitationJournal: mBio / Year: 2024
Title: Exploiting the Affimer platform against influenza A virus.
Authors: Oliver Debski-Antoniak / Alex Flynn / David P Klebl / Moisés H Rojas Rechy / Christian Tiede / Ian A Wilson / Stephen P Muench / Darren Tomlinson / Juan Fontana /
Abstract: Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the ...Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in , and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection . Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents.
IMPORTANCE: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short- ...IMPORTANCE: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection . We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.
History
DepositionJun 24, 2023-
Header (metadata) releaseJan 3, 2024-
Map releaseJan 3, 2024-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17724.png

Downloads & links

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Map

FileDownload / File: emd_17724.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 400 pix.
= 344. Å		0.86 Å/pix.
x 400 pix.
= 344. Å		0.86 Å/pix.
x 400 pix.
= 344. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.0018042236 - 2.2818851
Average (Standard dev.)0.00087233534 (±0.02228797)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 344.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_17724_additional_1.map
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Additional map: #2

Fileemd_17724_additional_2.map
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Half map: #1

Fileemd_17724_half_map_1.map
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Half map: #2

Fileemd_17724_half_map_2.map
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Sample components

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Entire : CryoEM reconstruction of hemagglutinin HK68 of Influenza A virus ...

EntireName: CryoEM reconstruction of hemagglutinin HK68 of Influenza A virus bound to an Affimer reagent
Components
  • Complex: CryoEM reconstruction of hemagglutinin HK68 of Influenza A virus bound to an Affimer reagent
    • Protein or peptide: Hemagglutinin HA2 chain
    • Protein or peptide: Affimer molecule (A31)
  • Protein or peptide: Hemagglutinin HA1 chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: CryoEM reconstruction of hemagglutinin HK68 of Influenza A virus ...

SupramoleculeName: CryoEM reconstruction of hemagglutinin HK68 of Influenza A virus bound to an Affimer reagent
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#3
Source (natural)Organism: Influenza A virus

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Macromolecule #1: Hemagglutinin HA1 chain

MacromoleculeName: Hemagglutinin HA1 chain / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 35.349754 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ADPGATLCLG HHAVPNGTLV KTITDDQIEV TNATELVQSS STGKICNNPH RILDGIDCTL IDALLGDPHC DVFQNETWDL FVERSKAFS NCYPYDVPDY ASLRSLVASS GTLEFITEGF TWTGVTQNGG SNACKRGPGS GFFSRLNWLT KSGSTYPVLN V TMPNNDNF ...String:
ADPGATLCLG HHAVPNGTLV KTITDDQIEV TNATELVQSS STGKICNNPH RILDGIDCTL IDALLGDPHC DVFQNETWDL FVERSKAFS NCYPYDVPDY ASLRSLVASS GTLEFITEGF TWTGVTQNGG SNACKRGPGS GFFSRLNWLT KSGSTYPVLN V TMPNNDNF DKLYIWGVHH PSTNQEQTSL YVQASGRVTV STRRSQQTII PNIGSRPWVR GLSSRISIYW TIVKPGDVLV IN SNGNLIA PRGYFKMRTG KSSIMRSDAP IDTCISECIT PNGSIPNDKP FQNVNKITYG ACPKYVKQNT LKLATGMRNV PEK QT

UniProtKB: Hemagglutinin

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Macromolecule #2: Hemagglutinin HA2 chain

MacromoleculeName: Hemagglutinin HA2 chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 20.21235 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GLFGAIAGFI ENGWEGMIDG WYGFRHQNSE GTGQAADLKS TQAAIDQING KLNRVIEKTN EKFHQIEKEF SEVEGRIQDL EKYVEDTKI DLWSYNAELL VALENQHTID LTDSEMNKLF EKTRRQLREN AEEMGNGCFK IYHKCDNACI ESIRNGTYDH D VYRDEALN NRFQIKG

UniProtKB: Hemagglutinin

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Macromolecule #3: Affimer molecule (A31)

MacromoleculeName: Affimer molecule (A31) / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.491819 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SLEIEELARF AVDEHNKKEN ALLEFVRVVK AKEQFNYDQW QDYTMYYLTL EAKDGGKKKL YEAKVWVKFV DSAFETENFK ELQEFKP

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 6 / 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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 56.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4fnk

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 104544
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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