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- EMDB-49373: CryoEM Structure of De Novo Antibody Fragment scFv 6 with C. diff... -

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Basic information

Entry
Database: EMDB / ID: EMD-49373
TitleCryoEM Structure of De Novo Antibody Fragment scFv 6 with C. difficile Toxin B (TcdB)
Map data
Sample
  • Complex: Toxin B in complex with scFv 6
    • Protein or peptide: Toxin B
  • Protein or peptide: scFv 6 Heavy Chain, scFv 6 Light Chain
KeywordsTcdB / De Novo / scFv 6 / De Novo Antibody / DE NOVO PROTEIN
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 speciessynthetic construct (others) / Clostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWeidle C / Borst AJ
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: 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, 2025-
Header (metadata) releaseNov 12, 2025-
Map releaseNov 12, 2025-
UpdateJan 7, 2026-
Current statusJan 7, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49373.png

Downloads & links

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Map

FileDownload / File: emd_49373.map.gz / Format: CCP4 / Size: 371.3 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.)
0.89 Å/pix.
x 460 pix.
= 407.1 Å		0.89 Å/pix.
x 460 pix.
= 407.1 Å		0.89 Å/pix.
x 460 pix.
= 407.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.885 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0339
Minimum - Maximum-0.12292408 - 0.24755934
Average (Standard dev.)-0.00001685791 (±0.00441912)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions460460460
Spacing460460460
CellA=B=C: 407.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_49373_additional_1.map
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Half map: #2

Fileemd_49373_half_map_1.map
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Half map: #1

Fileemd_49373_half_map_2.map
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Sample components

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Entire : Toxin B in complex with scFv 6

EntireName: Toxin B in complex with scFv 6
Components
  • Complex: Toxin B in complex with scFv 6
    • Protein or peptide: Toxin B
  • Protein or peptide: scFv 6 Heavy Chain, scFv 6 Light Chain

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Supramolecule #1: Toxin B in complex with scFv 6

SupramoleculeName: Toxin B in complex with scFv 6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
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.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 271.17119 KDa

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Macromolecule #1: Toxin B

MacromoleculeName: Toxin B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 241.229938 KDa
Recombinant expressionOrganism: Priestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
SequenceString: MYMSLVNRKQ LEKMANVRFR TQEDEYVAIL DALEEYHNMS ENTVVEKYLK LKDINSLTDI YIDTYKKSGR NKALKKFKEY LVTEVLELK NNNLTPVEKN LHFVAIGGQI NDTAINYINQ WKDVNSDYNV NVFYDSNAFL INTLKKTVVE SAINDTLESF R ENLNDPRF ...String:
MYMSLVNRKQ LEKMANVRFR TQEDEYVAIL DALEEYHNMS ENTVVEKYLK LKDINSLTDI YIDTYKKSGR NKALKKFKEY LVTEVLELK NNNLTPVEKN LHFVAIGGQI NDTAINYINQ WKDVNSDYNV NVFYDSNAFL INTLKKTVVE SAINDTLESF R ENLNDPRF DYNKFFRKRM EIIYDKQKNF INYYKAQREE NPELIIDDIV KTYLSNEYSK EIDELNTYIE ESLNKITQNS GN DVRNFEE FKNGESFNLY EQELVERWNL AAASDILRIS ALKEIGGMYL NVNMLPGIQP DLFESIEKPS SVTVDFWEMT KLE AIMKYK EYIPEYTSEH FDMLDEEVQS SFESVLASKS DKSEIFSSLG DMEASPLEVK IAFNSKGIIN QGLISVKDSY CSNL IVKQI ENRYKILNNS LNPAISEDND FNTTTNTFID SIMAEANADN GRFMMELGKY LRVGFFPDVK TTINLSGPEA YAAAY QDLL MFKEGSMNIH LIEADLRNFE ISKTNISQST EQEMASLWSF DDARAKAQFE EYKRNYFEGS AGEDDNLDFS QNIVVD KEY LLEKISSLAR SSERGYIHYI VQLQGDKISY EAACNLFAKT PYDSVLFQKN IEDSEIAYYY NPGDGEIQEI DKYKIPS II SDRPKIKLTF IGHGKDEFNT DIFAGFDVDS LSTEIEAAID LAKEDISPKS IEINLLGCNM FSYSINVEET YPGKLLLK V KDKISELMPS ISQDSIIVSA NQYEVRINSE GRRELLDHSG EWINKEESII KDISSKEYIS FNPKENKITV KSKNLPELS TLLQEIRNNS NSSDIELEEK VMLTECEINV ISNIDTQIVE ERIEEAKNLT SDSINYIKDE FKLIESISDA LCDLKQQNEL EDSHFISFE DISETDEGFS IRFINKETGE SIFVETEKTI FSEYANHITE EISKIKGTIF DTVNGKLVKK VNLDTTHEVN T LNAAFFIQ SLIEYNSSKE SLSNLSVAMK VQVYAQLFST GLNTITDAAK VVELVSTALD ETIDLLPTLS EGLPIIATII DG VSLGAAI KELSETSDPL LRQEIEAKIG IMAVNLTTAT TAIITSSLGI ASGFSILLVP LAGISAGIPS LVNNELVLRD KAT KVVDYF KHVSLVETEG VFTLLDDKIM MPQDDLVISE IDFNNNSIVL GKCEIWRMEG GSGHTVTDDI DHFFSAPSIT YREP HLSIY DVLEVQKEEL DLSKDLMVLP NAPNRVFAWE TGWTPGLRSL ENDGTKLLDR IRDNYEGEFY WRYFAFIADA LITTL KPRY EDTNIRINLD SNTRSFIVPI ITTEYIREKL SYSFYGSGGT YALSLSQYNM GINIELSESD VWIIDVDNVV RDVTIE SDK IKKGDLIEGI LSTLSIEENK IILNSHEINF SGEVNGSNGF VSLTFSILEG INAIIEVDLL SKSYKLLISG ELKILML NS NHIQQKIDYI GFNSELQKNI PYSFVDSEGK ENGFINGSTK EGLFVSELPD VVLISKVYMD DSKPSFGYYS NNLKDVKV I TKDNVNILTG YYLKDDIKIS LSLTLQDEKT IKLNSVHLDE SGVAEILKFM NRKGNTNTSD SLMSFLESMN IKSIFVNFL QSNIKFILDA NFIISGTTSI GQFEFICDEN DNIQPYFIKF NTLETNYTLY VGNRQNMIVE PNYDLDDSGD ISSTVINFSQ KYLYGIDSC VNKVVISPNI YTDEINITPV YETNNTYPEV IVLDANYINE KINVNINDLS IRYVWSNDGN DFILMSTSEE N KVSQVKIR FVNVFKDKTL ANKLSFNFSD KQDVPVSEII LSFTPSYYED GLIGYDLGLV SLYNEKFYIN NFGMMVSGLI YI NDSLYYF KPPVNNLITG FVTVGDDKYY FNPINGGAAS IGETIIDDKN YYFNQSGVLQ TGVFSTEDGF KYFAPANTLD ENL EGEAID FTGKLIIDEN IYYFDDNYRG AVEWKELDGE MHYFSPETGK AFKGLNQIGD YKYYFNSDGV MQKGFVSIND NKHY FDDSG VMKVGYTEID GKHFYFAENG EMQIGVFNTE DGFKYFAHHN EDLGNEEGEE ISYSGILNFN NKIYYFDDSF TAVVG WKDL EDGSKYYFDE DTAEAYIGGY RPHAGLRGSH HHHHH

UniProtKB: Toxin B

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Macromolecule #2: scFv 6 Heavy Chain, scFv 6 Light Chain

MacromoleculeName: scFv 6 Heavy Chain, scFv 6 Light Chain / type: protein_or_peptide / ID: 2 / Details: Heavy Chain with Linker to Light Chain / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 30.211605 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MGWSCIILFL VATATGVHSE VQLVESGGGL VQPGGSLRLS CAASGFSIKN TYIHWVRQAP GKGLEWVARI WPANGKTRYA DSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRQL DPYNLYGNDV WGQGTLVTVS SPNSASHSGS APQTSSAPGS D IQMTQSPS ...String:
MGWSCIILFL VATATGVHSE VQLVESGGGL VQPGGSLRLS CAASGFSIKN TYIHWVRQAP GKGLEWVARI WPANGKTRYA DSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRQL DPYNLYGNDV WGQGTLVTVS SPNSASHSGS APQTSSAPGS D IQMTQSPS SLSASVGDRV TITCKTSSSY VNWYQQKPGK APKLLIYRNS FRAPGVPSRF SGSRSGTDFT LTISSLQPED FA TYYCSTM NNDGNLVFGQ GTKVEIKENL YFQGSHHHHH H

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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

Concentration0.81 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: C-flat-2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 40 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 39.0 kPa / Details: 15 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 10897 / Average exposure time: 5.0 sec. / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination 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: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 3357543
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: 3D Ab Initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Non Uniform Refinement / Number images used: 19969
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Details: Non Uniform Refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Details: Non Uniform Refinement
Final 3D classificationNumber classes: 2 / Avg.num./class: 17141 / Details: 3D Classification
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

6oq5

source_name: PDB, initial_model_type: experimental modelTcdB
source_name: Other, initial_model_type: in silico modelDe Novo Design
DetailsTcdB 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9nfu:
CryoEM Structure of De Novo Antibody Fragment scFv 6 with C. difficile Toxin B (TcdB)

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