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- PDB-9nfu: CryoEM Structure of De Novo Antibody Fragment scFv 6 with C. diff... -

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Basic information

Entry
Database: PDB / ID: 9nfu
TitleCryoEM Structure of De Novo Antibody Fragment scFv 6 with C. difficile Toxin B (TcdB)
Components
  • Toxin B
  • scFv 6 Heavy Chain, scFv 6 Light Chain
KeywordsDE NOVO PROTEIN / TcdB / De Novo / scFv 6 / De Novo Antibody
Function / homology
Function and homology information


symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / glucosyltransferase activity / Transferases; Glycosyltransferases; Hexosyltransferases / host cell cytosol / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane ...symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / glucosyltransferase activity / Transferases; Glycosyltransferases; Hexosyltransferases / host cell cytosol / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis / extracellular region / metal ion binding / membrane
Similarity search - Function
TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain ...TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / Choline-binding repeat / Putative cell wall binding repeat / Cell wall/choline-binding repeat / Cell wall-binding repeat profile. / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Biological speciesClostridioides difficile (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWeidle, C. / Borst, A.J.
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: 2026
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 21, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toxin B
C: scFv 6 Heavy Chain, scFv 6 Light Chain


Theoretical massNumber of molelcules
Total (without water)271,4422
Polymers271,4422
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Toxin B


Mass: 241229.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: tcdB, toxB
Production host: Priestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
References: UniProt: P18177, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Antibody scFv 6 Heavy Chain, scFv 6 Light Chain


Mass: 30211.605 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Heavy Chain with Linker to Light Chain / Source: (gene. exp.) synthetic construct (others) / Production host: Cricetulus griseus (Chinese hamster)
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: Toxin B in complex with scFv 6 / Type: COMPLEX
Details: Toxin B was expressed and purified. scFv 6 was expressed and purified. scFv 6 was mixed with Toxin B at a 3:1 molar ratio.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.27117119 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
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.: 0.81 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA current / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 44 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10897
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3357543
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19969 / Algorithm: FOURIER SPACE / Details: Non Uniform Refinement / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: TcdB was built using the published 3.87 Angstrom crystal structure as a starting model (PDB: 6OQ5). The three bound VHH domains in the crystal structure were removed in PyMOL, and the ...Details: TcdB was built using the published 3.87 Angstrom crystal structure as a starting model (PDB: 6OQ5). The three bound VHH domains in the crystal structure were removed in PyMOL, and the structure was docked into density using Chimera. The initial model was refined in Coot before alignment with the design model in PyMOL, the scFv docked well in density. The entire model was refined with iterative rounds in Coot, Interactive Structure Optimization by Local Direct Exploration (ISOLDE) were performed at a simulated 25 Kelvin, and Phenix real-space refinement. The final model quality was analyzed using Molprobity.
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameType
16OQ5

6oq5

6OQ5TcdB1PDBexperimental model
2De Novo DesignOtherin silico model

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