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- PDB-9jec: Cryo-EM structure of canine TNF-alpha complexed with nanobody TNF30K -

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Basic information

Entry
Database: PDB / ID: 9jec
TitleCryo-EM structure of canine TNF-alpha complexed with nanobody TNF30K
Components
  • Tumor necrosis factor
  • nanobody TNF30K
KeywordsCYTOKINE / canine / TNF / nanobody / lymphokine
Function / homology
Function and homology information


TNFR1-induced proapoptotic signaling / Regulation of TNFR1 signaling / TNFR1-induced NF-kappa-B signaling pathway / TNFR1-mediated ceramide production / TNFR2 non-canonical NF-kB pathway / TNF signaling / regulation of multicellular organismal process / regulation of biological quality / regulation of secretion / negative regulation of protein-containing complex disassembly ...TNFR1-induced proapoptotic signaling / Regulation of TNFR1 signaling / TNFR1-induced NF-kappa-B signaling pathway / TNFR1-mediated ceramide production / TNFR2 non-canonical NF-kB pathway / TNF signaling / regulation of multicellular organismal process / regulation of biological quality / regulation of secretion / negative regulation of protein-containing complex disassembly / vascular endothelial growth factor production / necroptotic signaling pathway / positive regulation of protein-containing complex disassembly / tumor necrosis factor receptor binding / positive regulation of extrinsic apoptotic signaling pathway / regulation of developmental process / positive regulation of JUN kinase activity / positive regulation of MAP kinase activity / extrinsic apoptotic signaling pathway via death domain receptors / cytokine activity / tumor necrosis factor-mediated signaling pathway / positive regulation of NF-kappaB transcription factor activity / positive regulation of protein phosphorylation / positive regulation of canonical NF-kappaB signal transduction / immune response / positive regulation of apoptotic process / cell surface / positive regulation of transcription by RNA polymerase II / extracellular space / plasma membrane
Similarity search - Function
Tumour necrosis factor alpha / Tumour necrosis factor / 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
Tumor necrosis factor
Similarity search - Component
Biological speciesCanis lupus familiaris (dog)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWeng, J. / Li, Z. / Xu, J. / Wei, Z.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32000032 China
Other governmentSKLBEE2022007
CitationJournal: To Be Published
Title: Cryo-EM structure of canine TNF-alpha complexed with nanobody TNF30K
Authors: Weng, J. / Li, Z. / Xu, J. / Wei, Z.
History
DepositionSep 3, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 24, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tumor necrosis factor
B: Tumor necrosis factor
C: Tumor necrosis factor
D: nanobody TNF30K
E: nanobody TNF30K
F: nanobody TNF30K


Theoretical massNumber of molelcules
Total (without water)100,7616
Polymers100,7616
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 Tumor necrosis factor / Cachectin / TNF-alpha / Tumor necrosis factor ligand superfamily member 2 / TNF-a


Mass: 19123.730 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Canis lupus familiaris (dog) / Gene: TNF, TNFA, TNFSF2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P51742
#2: Antibody nanobody TNF30K


Mass: 14463.364 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21(DE3) (bacteria)
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: Hexamer complex of canine TNF-alpha and nanobody TNF30K
Type: COMPLEX
Details: a hexamer complex consist of one canine TNF-alpha trimer with three nanobody TNF30K molecules
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.01 MDa / Experimental value: NO
Source (natural)Organism: Canis lupus familiaris (dog)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.4
Details: 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mmol/LDisodium hydrogen phosphateNa2HPO41
21.8 mmol/LPotassium dihydrogen phosphateKH2PO41
3137 mmol/Lsodium chlorideNaCl1
42.7 mmol/Lpotassium chlorideKCl1
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was FPLC purified and monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 543326 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 104.38 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00556120
ELECTRON MICROSCOPYf_angle_d0.63658313
ELECTRON MICROSCOPYf_chiral_restr0.0468921
ELECTRON MICROSCOPYf_plane_restr0.00431068
ELECTRON MICROSCOPYf_dihedral_angle_d4.4566837

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