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- EMDB-61412: Cryo-EM structure of canine TNF-alpha complexed with nanobody TNF30K -

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Basic information

Entry
Database: EMDB / ID: EMD-61412
TitleCryo-EM structure of canine TNF-alpha complexed with nanobody TNF30K
Map data
Sample
  • Complex: Hexamer complex of canine TNF-alpha and nanobody TNF30K
    • Protein or peptide: Tumor necrosis factor
    • Protein or peptide: nanobody TNF30K
Keywordscanine / TNF / nanobody / lymphokine / CYTOKINE
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 / negative regulation of branching involved in lung morphogenesis / positive regulation of interleukin-33 production / positive regulation of blood microparticle formation / positive regulation of chronic inflammatory response to antigenic stimulus ...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 / negative regulation of branching involved in lung morphogenesis / positive regulation of interleukin-33 production / positive regulation of blood microparticle formation / positive regulation of chronic inflammatory response to antigenic stimulus / positive regulation of protein transport / positive regulation of leukocyte adhesion to arterial endothelial cell / positive regulation of vitamin D biosynthetic process / positive regulation of translational initiation by iron / regulation of membrane lipid metabolic process / regulation of endothelial cell apoptotic process / chronic inflammatory response to antigenic stimulus / regulation of branching involved in salivary gland morphogenesis / negative regulation of protein-containing complex disassembly / positive regulation of humoral immune response mediated by circulating immunoglobulin / positive regulation of hair follicle development / negative regulation of vascular wound healing / negative regulation of cytokine production involved in immune response / negative regulation of amyloid-beta clearance / inflammatory response to wounding / death receptor agonist activity / epithelial cell proliferation involved in salivary gland morphogenesis / embryonic digestive tract development / negative regulation of D-glucose import / vascular endothelial growth factor production / positive regulation of fever generation / positive regulation of synoviocyte proliferation / positive regulation of calcineurin-NFAT signaling cascade / necroptotic signaling pathway / leukocyte tethering or rolling / negative regulation of myoblast differentiation / positive regulation of protein-containing complex disassembly / regulation of establishment of endothelial barrier / endothelial cell apoptotic process / positive regulation of protein localization to cell surface / macrophage activation involved in immune response / positive regulation of osteoclast differentiation / tumor necrosis factor receptor binding / positive regulation of heterotypic cell-cell adhesion / positive regulation of macrophage derived foam cell differentiation / positive regulation of cytokine production involved in inflammatory response / regulation of immunoglobulin production / negative regulation of mitotic cell cycle / positive regulation of podosome assembly / positive regulation of membrane protein ectodomain proteolysis / positive regulation of chemokine (C-X-C motif) ligand 2 production / positive regulation of extrinsic apoptotic signaling pathway / negative regulation of bicellular tight junction assembly / regulation of reactive oxygen species metabolic process / positive regulation of amyloid-beta formation / negative regulation of viral genome replication / regulation of synapse organization / negative regulation of endothelial cell proliferation / positive regulation of JUN kinase activity / positive regulation of MAP kinase activity / negative regulation of interleukin-6 production / humoral immune response / negative regulation of blood vessel endothelial cell migration / positive regulation of glial cell proliferation / extrinsic apoptotic signaling pathway via death domain receptors / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / negative regulation of osteoblast differentiation / positive regulation of synaptic transmission / phagocytic cup / negative regulation of lipid catabolic process / positive regulation of vascular associated smooth muscle cell proliferation / JNK cascade / extracellular matrix organization / antiviral innate immune response / regulation of insulin secretion / response to glucocorticoid / osteoclast differentiation / positive regulation of interleukin-1 beta production / cytokine activity / tumor necrosis factor-mediated signaling pathway / cellular response to ionizing radiation / positive regulation of interleukin-8 production / protein localization to plasma membrane / positive regulation of protein localization to plasma membrane / positive regulation of JNK cascade / cellular response to amino acid stimulus / phosphatidylinositol 3-kinase/protein kinase B signal transduction / microglial cell activation / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of NF-kappaB transcription factor activity / recycling endosome / cellular response to type II interferon / cellular response to nicotine / positive regulation of interleukin-6 production / positive regulation of miRNA transcription / positive regulation of type II interferon production / positive regulation of protein phosphorylation / glucose metabolic process / intrinsic apoptotic signaling pathway in response to DNA damage / positive regulation of nitric oxide biosynthetic process
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWeng J / Li Z / Xu J / Wei Z
Funding support China, 2 items
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, 2024-
Header (metadata) releaseSep 24, 2025-
Map releaseSep 24, 2025-
UpdateSep 24, 2025-
Current statusSep 24, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61412.png

Downloads & links

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Map

FileDownload / File: emd_61412.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 180 pix.
= 153. Å		0.85 Å/pix.
x 180 pix.
= 153. Å		0.85 Å/pix.
x 180 pix.
= 153. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.574381 - 2.2202692
Average (Standard dev.)-0.0013610445 (±0.092956275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 153.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_61412_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_61412_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexamer complex of canine TNF-alpha and nanobody TNF30K

EntireName: Hexamer complex of canine TNF-alpha and nanobody TNF30K
Components
  • Complex: Hexamer complex of canine TNF-alpha and nanobody TNF30K
    • Protein or peptide: Tumor necrosis factor
    • Protein or peptide: nanobody TNF30K

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Supramolecule #1: Hexamer complex of canine TNF-alpha and nanobody TNF30K

SupramoleculeName: Hexamer complex of canine TNF-alpha and nanobody TNF30K
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: a hexamer complex consist of one canine TNF-alpha trimer with three nanobody TNF30K molecules
Source (natural)Organism: Canis lupus familiaris (dog)
Molecular weightTheoretical: 1.01 MDa

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Macromolecule #1: Tumor necrosis factor

MacromoleculeName: Tumor necrosis factor / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Canis lupus familiaris (dog)
Molecular weightTheoretical: 19.12373 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MKKLLIAAGS VKSSSRTPSD KPVAHVVANP EAEGQLQWLS RRANALLANG VELTDNQLIV PSDGLYLIYS QVLFKGQGCP STHVLLTHT ISRFAVSYQT KVNLLSAIKS PCQRETPEGT EAKPWYEPIY LGGVFQLEKG DRLSAEINLP NYLDFAESGQ V YFGIIALH HHHHH

UniProtKB: Tumor necrosis factor

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Macromolecule #2: nanobody TNF30K

MacromoleculeName: nanobody TNF30K / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.463364 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MKKLLIAAGS EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMYWVRQA PGKGLEWVSK INTNGLITKY PDSVKGRFTI SRDNAKNTL YLQMNSLRPE DTAVYYCARS PSGFNRGQGT LVTVSSHHHH HH

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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

Concentration1.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mmol/LNa2HPO4Disodium hydrogen phosphate
1.8 mmol/LKH2PO4Potassium dihydrogen phosphate
137.0 mmol/LNaClsodium chloride
2.7 mmol/LKClpotassium chloride

Details: 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was FPLC purified and monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 2.5 sec. / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 543326
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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