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- PDB-9nh7: CryoEM Structure of De Novo VHH, VHH_flu_01, bound to influenza H... -

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Basic information

Entry
Database: PDB / ID: 9nh7
TitleCryoEM Structure of De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1.
Components
  • (Hemagglutinin ...) x 2
  • VHH_flu_01
KeywordsDE NOVO PROTEIN / Influenza / Flu / HA / hemagglutinin / H1N1 / antibody / De Novo / protein design / VHH_flu_01 / De Novo Antibody / 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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsBorst, A.J. / Weidle, C.
Funding support United States, European Union, United Kingdom, 11items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release
Revision 1.1Nov 19, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: VHH_flu_01
A: Hemagglutinin HA1 chain
B: Hemagglutinin HA1 chain
C: Hemagglutinin HA1 chain
F: VHH_flu_01
G: Hemagglutinin HA2 chain
H: Hemagglutinin HA2 chain
I: Hemagglutinin HA2 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,71726
Polymers217,7068
Non-polymers6,01218
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Hemagglutinin ... , 2 types, 6 molecules ABCGHI

#2: Protein Hemagglutinin HA1 chain


Mass: 35923.340 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/USA:Iowa/1943 H1N1)
Strain: A/USA:Iowa/1943 H1N1 / Gene: HA / Production host: Homo sapiens (human) / References: UniProt: A4GCK8
#3: Protein Hemagglutinin HA2 chain


Mass: 26623.463 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/USA:Iowa/1943 H1N1)
Strain: A/USA:Iowa/1943 H1N1 / Gene: HA / Production host: Homo sapiens (human) / References: UniProt: A4GCK8

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Antibody , 1 types, 2 molecules EF

#1: Antibody VHH_flu_01


Mass: 15032.718 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Sugars , 3 types, 18 molecules

#4: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestY
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: De Novo VHH, VHH_flu_01, bound to influenza HA, strain A/USA:Iowa/1943 H1N1.
Type: COMPLEX
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.
Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.09244733 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: 150 mM NaCl, 40 mM Tris/ HCl pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
240 nMtris(hydroxymethyl)aminomethane/Hydrochloric AcidTris/HCl1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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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 magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16954
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIX1.21.2_5419model refinement
14PyMOLmodel refinement
15Cootmodel refinement
16ISOLDEmodel refinement
17UCSF ChimeraXmodel refinement
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5869679
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 288441 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameType
18SK7

8sk7

8SK7Trimeric influenza HA glycoprotein (strain A/USA:Iowa/1943 H1N11PDBexperimental model
2De Novo DesignOtherin silico model
RefinementHighest resolution: 3.02 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413249
ELECTRON MICROSCOPYf_angle_d0.56917990
ELECTRON MICROSCOPYf_dihedral_angle_d12.8414696
ELECTRON MICROSCOPYf_chiral_restr0.0492053
ELECTRON MICROSCOPYf_plane_restr0.0042300

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