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- EMDB-52634: Single particle cryo electron microscopy of a Fab fragment bound ... -

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Basic information

Entry
Database: EMDB / ID: EMD-52634
TitleSingle particle cryo electron microscopy of a Fab fragment bound to recombinant human CD40 ligand
Map dataSymmetry expanded focus map (sharpened) of CD40 ligand with Fab fragment bound
Sample
  • Complex: Incubated sample containing Fab fragment and recombinant human CD40 ligand.
    • Protein or peptide: Fab20 heavy chain
    • Protein or peptide: Fab20 light chain
    • Protein or peptide: CD40 ligand, soluble form
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsCD40 ligand inhibitor / CD154 inhibitor / autoimmunity / Fab fragment / IMMUNE SYSTEM
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 / : / integrin-mediated signaling pathway / protein serine/threonine kinase activator activity / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKristoffersen EL / Schinkel T / Andersen ES
Funding support Denmark, 1 items
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, 2025-
Header (metadata) releaseFeb 25, 2026-
Map releaseFeb 25, 2026-
UpdateMar 11, 2026-
Current statusMar 11, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52634.png

Downloads & links

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Map

FileDownload / File: emd_52634.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetry expanded focus map (sharpened) of CD40 ligand with Fab fragment bound
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.29 Å/pix.
x 300 pix.
= 388.2 Å		1.29 Å/pix.
x 300 pix.
= 388.2 Å		1.29 Å/pix.
x 300 pix.
= 388.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.294 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.696242 - 2.8209622
Average (Standard dev.)-0.0000765345 (±0.025903292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 388.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52634_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Symmetry expanded focus map (half map B) of...

Fileemd_52634_half_map_1.map
AnnotationSymmetry expanded focus map (half map B) of CD40 ligand with Fab fragment bound
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Symmetry expanded focus map (half map A) of...

Fileemd_52634_half_map_2.map
AnnotationSymmetry expanded focus map (half map A) of CD40 ligand with Fab fragment bound
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Incubated sample containing Fab fragment and recombinant human CD...

EntireName: Incubated sample containing Fab fragment and recombinant human CD40 ligand.
Components
  • Complex: Incubated sample containing Fab fragment and recombinant human CD40 ligand.
    • Protein or peptide: Fab20 heavy chain
    • Protein or peptide: Fab20 light chain
    • Protein or peptide: CD40 ligand, soluble form
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Incubated sample containing Fab fragment and recombinant human CD...

SupramoleculeName: Incubated sample containing Fab fragment and recombinant human CD40 ligand.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Buffer: 1x PBS, 1 mM MgCl2; 1:3 molar ratio, 2.47 uM rhCD40L and 7.42 uM Fab20
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Fab20 heavy chain

MacromoleculeName: Fab20 heavy chain / type: protein_or_peptide / ID: 1 / Details: Fab fragment heavy chain, only the variable region / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 12.772167 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT GYGVHWVRQS PGKGLEWLGM IWSGGSTDYN AAFISRLSIS KDSSKSQVFF KMNSLQAND TAIYYCARSS GNYGEDAMDY WGQGTSVTV

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Macromolecule #2: Fab20 light chain

MacromoleculeName: Fab20 light chain / type: protein_or_peptide / ID: 2 / Details: Fab fragment light chain, only the variable region / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.825098 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
ETTVTQSPAS LSMAIGEKVT IRCITSTDID DDMNWYQQKP GEPPELLISE DNTLRPGVPS RFSSSGYGTD FVFAIENMLP EDVADYFCL QSDNLPFTFG SGTKLEIK

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Macromolecule #3: CD40 ligand, soluble form

MacromoleculeName: CD40 ligand, soluble form / type: protein_or_peptide / ID: 3
Details: One subunit of extracellular domain of trimeric hCD40L
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.512547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
NPQIAAHVIS EASSKTTSVL QWAEKGYYTM SNNLVTLENG KQLTVKRQGL YYIYAQVTFC SNREASSQAP FIASLCLKSP GRFERILLR AANTHSSAKP CGQQSIHLGG VFELQPGASV FVNVTDPSQV SHGTGFTSFG LLKL

UniProtKB: CD40 ligand

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 13 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7.5
Component:
ConcentrationFormulaName
1.0 xPBSPhosphate-buffered saline
1.0 mMMgCl2magnesium chloride
GridModel: Au-flat 1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.015 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 5673 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Details: 1680 movies recorded from one grid and 3993 movies recorded from other grid. After curation 4307 were used.
Electron beamAcceleration voltage: 300 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: 130000
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

CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 311649
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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