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- PDB-9i5n: Single particle cryo electron microscopy of a Fab fragment bound ... -

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Basic information

Entry
Database: PDB / ID: 9i5n
TitleSingle particle cryo electron microscopy of a Fab fragment bound to recombinant human CD40 ligand
Components
  • CD40 ligand, soluble form
  • Fab20 heavy chain
  • Fab20 light chain
KeywordsIMMUNE SYSTEM / CD40 ligand inhibitor / CD154 inhibitor / autoimmunity / Fab fragment
Function / homology
Function and homology information


CD40 receptor binding / TNF receptor superfamily (TNFSF) members mediating non-canonical NF-kB pathway / CD40 signaling pathway / regulation of immunoglobulin production / isotype switching / tumor necrosis factor receptor binding / positive regulation of extrinsic apoptotic signaling pathway / leukocyte cell-cell adhesion / positive regulation of interleukin-4 production / B cell proliferation ...CD40 receptor binding / TNF receptor superfamily (TNFSF) members mediating non-canonical NF-kB pathway / CD40 signaling pathway / regulation of immunoglobulin production / isotype switching / tumor necrosis factor receptor binding / positive regulation of extrinsic apoptotic signaling pathway / leukocyte cell-cell adhesion / positive regulation of interleukin-4 production / B cell proliferation / positive regulation of interleukin-10 production / positive regulation of endothelial cell apoptotic process / T cell costimulation / positive regulation of interleukin-12 production / positive regulation of T cell proliferation / B cell differentiation / cytokine activity / : / protein serine/threonine kinase activator activity / integrin-mediated signaling pathway / TNFR2 non-canonical NF-kB pathway / platelet activation / integrin binding / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / positive regulation of canonical NF-kappaB signal transduction / cell surface receptor signaling pathway / inflammatory response / external side of plasma membrane / negative regulation of apoptotic process / cell surface / Golgi apparatus / : / membrane / plasma membrane
Similarity search - Function
CD40 ligand / Tumour necrosis factor, conserved site / Tumor necrosis factor (TNF) homology domain (THD) signature. / Tumour necrosis factor family. / TNF(Tumour Necrosis Factor) family / Tumour necrosis factor domain / Tumor necrosis factor (TNF) homology domain (THD) profile. / Tumour necrosis factor-like domain superfamily
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKristoffersen, E.L. / Schinkel, T. / Andersen, E.S.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF21OC0070452 Denmark
CitationJournal: Eur J Immunol / Year: 2026
Title: Curbing Autoimmunity: A New Fab Fragment Targeting CD40-CD40L Halts B-Cell Activation and Differentiation.
Authors: Kathrine Pedersen / Kenneth Green / Emil L Kristoffersen / Thea Schinkel / Annette G Hansen / Yaseelan Palarasah / Anne B Rovsing / Ebbe S Andersen / Julián Valero / Laia Civit / Nick S ...Authors: Kathrine Pedersen / Kenneth Green / Emil L Kristoffersen / Thea Schinkel / Annette G Hansen / Yaseelan Palarasah / Anne B Rovsing / Ebbe S Andersen / Julián Valero / Laia Civit / Nick S Laursen / Anne Troldborg / Søren E Degn / Steffen Thiel /
Abstract: Dysregulation of the CD40-CD40L axis is implicated in autoimmune diseases. Early clinical trials targeting CD40L with antibodies failed due to Fc-mediated side effects. To address this, we developed ...Dysregulation of the CD40-CD40L axis is implicated in autoimmune diseases. Early clinical trials targeting CD40L with antibodies failed due to Fc-mediated side effects. To address this, we developed an anti-CD40L Fab fragment, Fab20, designed to block B-cell activation. Fab20 was evaluated for its binding properties, CD40-CD40L inhibition, and effects on human B-cell activation and differentiation using immunoassays, cryo-electron microscopy, flow cytometry, and cell cultures. Fab20 binds CD40L with a dissociation constant of 70 nM. Structural analysis revealed a "propeller-like" structure consisting of three Fabs binding to the CD40L trimer, sterically blocking parts of the CD40 binding site. Fab20 effectively inhibited B-cell activation, maintaining naïve B cells in their inactive state, and suppressed antibody (IgG) production over 14 days. Fab20 represents a promising novel therapeutic approach for treating autoimmune diseases driven by CD40-CD40L dysregulation. Its mechanism of action, coupled with the absence of Fc-mediated effects, suggests a favorable safety profile.
History
DepositionJan 28, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 25, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 11, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Mar 11, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _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
B: Fab20 heavy chain
A: Fab20 light chain
C: CD40 ligand, soluble form
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,1344
Polymers40,1103
Non-polymers241
Water23413
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

#1: Antibody Fab20 heavy chain


Mass: 12772.167 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Fab fragment heavy chain, only the variable region / Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Antibody Fab20 light chain


Mass: 11825.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Fab fragment light chain, only the variable region / Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#3: Protein CD40 ligand, soluble form / sCD40L


Mass: 15512.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: One subunit of extracellular domain of trimeric hCD40L
Source: (gene. exp.) Homo sapiens (human) / Gene: CD40LG, CD40L, TNFSF5, TRAP / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P29965
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 13 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: Incubated sample containing Fab fragment and recombinant human CD40 ligand.
Type: COMPLEX
Details: Buffer: 1x PBS, 1 mM MgCl2; 1:3 molar ratio, 2.47 uM rhCD40L and 7.42 uM Fab20
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
11 xPhosphate-buffered salinePBS1
21 mMmagnesium chlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 277 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: 130000 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: 1.5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5673
Details: 1680 movies recorded from one grid and 3993 movies recorded from other grid. After curation 4307 were used.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.6.0particle selectionTopaz
2EPUimage acquisition
4cryoSPARC4.6.0CTF correction
7ISOLDEmodel fitting
13PHENIX1.21_5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 311649 / Symmetry type: POINT
RefinementHighest resolution: 3.4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0032881
ELECTRON MICROSCOPYf_angle_d0.4843910
ELECTRON MICROSCOPYf_dihedral_angle_d7.331406
ELECTRON MICROSCOPYf_chiral_restr0.04434
ELECTRON MICROSCOPYf_plane_restr0.004507

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