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- EMDB-49405: CryoEM Structure of De Novo VHH, VHH_flu_01, bound to influenza H... -

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Basic information

Entry
Database: EMDB / ID: EMD-49405
TitleCryoEM Structure of De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1
Map dataDeepEMhancer
Sample
  • Complex: De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1.
    • Protein or peptide: VHH_flu_01
    • Protein or peptide: Hemagglutinin HA1 chain
    • Protein or peptide: Hemagglutinin HA2 chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsInfluenza / Flu / HA / hemagglutinin / H1N1 / antibody / De Novo / protein design / VHH_flu_01 / De Novo Antibody / DE NOVO PROTEIN / RFdiffusion / RFantibody
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
Biological speciessynthetic construct (others) / Influenza A virus (strain A/USA:Iowa/1943 H1N1)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsBorst AJ / Weidle C
Funding support United States, European Union, United Kingdom, 11 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Department of Energy (DOE, United States)DE-SC0018940 MOD03 United States
National Institutes of Health/National Eye Institute (NIH/NEI)T32EY032448 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA260415 United States
Bill & Melinda Gates FoundationINV-010680 United States
European Molecular Biology Organization (EMBO)ALTF 292-2022European Union
Defense Threat Reduction Agency (DTRA)HDTRA1-21-1-0007 United States
National Science Foundation (NSF, United States)NERSC award BER-ERCAP0022018 United States
Cancer Research UKCGCATF-2021/100002 United Kingdom
National Institutes of Health/National Cancer Institute (NIH/NCI)CA278687-01 United States
Defense Threat Reduction Agency (DTRA)HDTRA1-21-1-0038 United States
CitationJournal: Nature / Year: 2025
Title: Atomically accurate de novo design of antibodies with RFdiffusion.
Authors: Nathaniel R Bennett / Joseph L Watson / Robert J Ragotte / Andrew J Borst / DéJenaé L See / Connor Weidle / Riti Biswas / Yutong Yu / Ellen L Shrock / Russell Ault / Philip J Y Leung / ...Authors: Nathaniel R Bennett / Joseph L Watson / Robert J Ragotte / Andrew J Borst / DéJenaé L See / Connor Weidle / Riti Biswas / Yutong Yu / Ellen L Shrock / Russell Ault / Philip J Y Leung / Buwei Huang / Inna Goreshnik / John Tam / Kenneth D Carr / Benedikt Singer / Cameron Criswell / Basile I M Wicky / Dionne Vafeados / Mariana Garcia Sanchez / Ho Min Kim / Susana Vázquez Torres / Sidney Chan / Shirley M Sun / Timothy T Spear / Yi Sun / Keelan O'Reilly / John M Maris / Nikolaos G Sgourakis / Roman A Melnyk / Chang C Liu / David Baker /
Abstract: Despite the central role of antibodies in modern medicine, no method currently exists to design novel, epitope-specific antibodies entirely in silico. Instead, antibody discovery currently relies on ...Despite the central role of antibodies in modern medicine, no method currently exists to design novel, epitope-specific antibodies entirely in silico. Instead, antibody discovery currently relies on immunization, random library screening or the isolation of antibodies directly from patients. Here we demonstrate that combining computational protein design using a fine-tuned RFdiffusion network with yeast display screening enables the de novo generation of antibody variable heavy chains (VHHs), single-chain variable fragments (scFvs) and full antibodies that bind to user-specified epitopes with atomic-level precision. We experimentally characterize VHH binders to four disease-relevant epitopes. Cryo-electron microscopy confirms the binding pose of designed VHHs targeting influenza haemagglutinin and Clostridium difficile toxin B (TcdB). A high-resolution structure of the influenza-targeting VHH confirms atomic accuracy of the designed complementarity-determining regions (CDRs). Although initial computational designs exhibit modest affinity (tens to hundreds of nanomolar K), affinity maturation using OrthoRep enables production of single-digit nanomolar binders that maintain the intended epitope selectivity. We further demonstrate the de novo design of scFvs to TcdB and a PHOX2B peptide-MHC complex by combining designed heavy-chain and light-chain CDRs. Cryo-electron microscopy confirms the binding pose for two distinct TcdB scFvs, with high-resolution data for one design verifying the atomically accurate design of the conformations of all six CDR loops. Our approach establishes a framework for the computational design, screening and characterization of fully de novo antibodies with atomic-level precision in both structure and epitope targeting.
History
DepositionFeb 24, 2025-
Header (metadata) releaseNov 12, 2025-
Map releaseNov 12, 2025-
UpdateNov 19, 2025-
Current statusNov 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49405.png

Downloads & links

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Map

FileDownload / File: emd_49405.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 340 pix.
= 285.6 Å		0.84 Å/pix.
x 340 pix.
= 285.6 Å		0.84 Å/pix.
x 340 pix.
= 285.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00909
Minimum - Maximum-0.028199285 - 1.6458527
Average (Standard dev.)0.0010399796 (±0.021002438)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 285.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened

Fileemd_49405_additional_1.map
AnnotationUnsharpened
Projections & Slices
AxesZYX

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Additional map: Sharpened

Fileemd_49405_additional_2.map
AnnotationSharpened
Projections & Slices
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Half map: Half Map B

Fileemd_49405_half_map_1.map
AnnotationHalf Map B
Projections & Slices
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Histogram

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Half map: Half Map A

Fileemd_49405_half_map_2.map
AnnotationHalf Map A
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Sample components

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Entire : De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa...

EntireName: De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1.
Components
  • Complex: De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1.
    • Protein or peptide: VHH_flu_01
    • Protein or peptide: Hemagglutinin HA1 chain
    • Protein or peptide: Hemagglutinin HA2 chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa...

SupramoleculeName: De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Influenza HA, strain A/USA:Iowa/1943 H1N1 was expressed and purified. VHH_flu_01 was expressed and purified. VHH_flu_01 was mixed with Influenza HA, strain A/USA:Iowa/1943 H1N1 at a 3:1 molar ratio.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 92.44733 KDa

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Macromolecule #1: VHH_flu_01

MacromoleculeName: VHH_flu_01 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.032718 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGQVQLVES GGGLVQPGGS LRLSCAASGK YVNLMSLGWF RQAPGQGLEA VAAISFDGKK TYYADSVKGR FTISRDNSKN TLYLQMNSL RAEDTAVYYC AASVVDSLGV GFYSYWGQGT LVTVSGSGSH HWGSTHHHHH H

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Macromolecule #2: Hemagglutinin HA1 chain

MacromoleculeName: Hemagglutinin HA1 chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (strain A/USA:Iowa/1943 H1N1) / Strain: A/USA:Iowa/1943 H1N1
Molecular weightTheoretical: 35.92334 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DTICIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDSHNGK LCRLKGIAPL QLGKCNIAGW ILGNPECESL LSERSWSYIV ETPNSENGT CFPGDFIDYE ELREQLSSVS SFERFEIFSK ESSWPKHTTG GVTAACSHAG KSSFYRNLLW LTEKDGSYPN L NNSYVNKK ...String:
DTICIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDSHNGK LCRLKGIAPL QLGKCNIAGW ILGNPECESL LSERSWSYIV ETPNSENGT CFPGDFIDYE ELREQLSSVS SFERFEIFSK ESSWPKHTTG GVTAACSHAG KSSFYRNLLW LTEKDGSYPN L NNSYVNKK GKEVLVLWGV HHPSNIKDQQ TLYQKENAYV SVVSSNYNRR FTPEIAERPK VRGQAGRINY YWTLLKPGDT IM FEANGNL IAPWYAFALS RGFGSGIITS NASMHECDTK CQTPQGAINS SLPFQNIHPI TIGECPKYVR STKLRMVTGL RNI P

UniProtKB: Hemagglutinin

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Macromolecule #3: Hemagglutinin HA2 chain

MacromoleculeName: Hemagglutinin HA2 chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (strain A/USA:Iowa/1943 H1N1) / Strain: A/USA:Iowa/1943 H1N1
Molecular weightTheoretical: 26.623463 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SIQSRGLFGA IAGFIEGGWT GMIDGWYGYH WQNEQGSGYA ADQKSTQNAI NGITNIVNSV IEKMNTQFTA VGKEFNNLEK RMENLNKKV DDGFLDIWTY NAELLVLLIN ERTLDFHDSN VKNLYEKVKN QLRNNAKEIG NGCFEFYHKC NNECMESVKN G TYDYPKYS ...String:
SIQSRGLFGA IAGFIEGGWT GMIDGWYGYH WQNEQGSGYA ADQKSTQNAI NGITNIVNSV IEKMNTQFTA VGKEFNNLEK RMENLNKKV DDGFLDIWTY NAELLVLLIN ERTLDFHDSN VKNLYEKVKN QLRNNAKEIG NGCFEFYHKC NNECMESVKN G TYDYPKYS EESKLNREKI DGSGYIPEAP RDGQAYVRKD GEWVLLSTFL GSGLNDIFEA QKIEWHEGHH HHHH

UniProtKB: Hemagglutinin

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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: 12 / 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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
40.0 nMTris/HCltris(hydroxymethyl)aminomethane/Hydrochloric Acid

Details: 150 mM NaCl, 40 mM Tris/ HCl pH 7.5
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 16954 / Average exposure time: 5.0 sec. / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
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: 5869679
CTF correctionSoftware - Name: cryoSPARC / Details: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: 3D Ab Initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 288441
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Non Uniform Refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Non Uniform Refinement
Final 3D classificationNumber classes: 2 / Avg.num./class: 17141 / Software - Name: cryoSPARC / Details: 3D Classification
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8sk7

source_name: PDB, initial_model_type: experimental modelTrimeric influenza HA glycoprotein (strain A/USA:Iowa/1943 H1N1
source_name: Other, initial_model_type: in silico modelDe Novo Design
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9nh7:
CryoEM Structure of De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1.

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