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- PDB-21tw: Cryo-EM structure of TNF-alpha in complex with two anti-TNF-alpha... -

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Basic information

Entry
Database: PDB / ID: 21tw
TitleCryo-EM structure of TNF-alpha in complex with two anti-TNF-alpha nanobodies, TNF30, derived from the TNF-alpha inhibitor Ozoralizumab (OZR)
Components
  • Tumor necrosis factor
  • anti-TNF-alpha nanobodies, TNF30, derived from Ozoralizumab (OZR)
KeywordsCYTOKINE / NANOBODY / VHH / TNF-alpha / HSA / Ozoralizumab / OZR
Function / homology
Function and homology information


response to Gram-negative bacterium / negative regulation of L-glutamate import across plasma membrane / negative regulation of bile acid secretion / positive regulation of interleukin-33 production / positive regulation of neutrophil activation / negative regulation of branching involved in lung morphogenesis / positive regulation of fractalkine production / positive regulation of blood microparticle formation / response to 3,3',5-triiodo-L-thyronine / positive regulation of protein transport ...response to Gram-negative bacterium / negative regulation of L-glutamate import across plasma membrane / negative regulation of bile acid secretion / positive regulation of interleukin-33 production / positive regulation of neutrophil activation / negative regulation of branching involved in lung morphogenesis / positive regulation of fractalkine production / positive regulation of blood microparticle formation / response to 3,3',5-triiodo-L-thyronine / positive regulation of protein transport / positive regulation of vitamin D biosynthetic process / chronic inflammatory response to antigenic stimulus / regulation of endothelial cell apoptotic process / response to macrophage colony-stimulating factor / negative regulation of myelination / positive regulation of leukocyte adhesion to arterial endothelial cell / response to gold nanoparticle / response to quercetin / Differentiation of naive CD4+ T cells to T helper 1 cells (Th1 cells) / negative regulation of vascular wound healing / negative regulation of amyloid-beta clearance / negative regulation of cytokine production involved in immune response / positive regulation of podosome assembly / positive regulation of hair follicle development / inflammatory response to wounding / response to resveratrol / positive regulation of interleukin-18 production / positive regulation of hepatocyte proliferation / positive regulation of action potential / toll-like receptor 3 signaling pathway / TNF signaling / negative regulation of D-glucose import across plasma membrane / embryonic digestive tract development / vascular endothelial growth factor production / positive regulation of fever generation / positive regulation of calcineurin-NFAT signaling cascade / response to fructose / positive regulation of protein localization to cell surface / negative regulation of mitotic cell cycle / leukocyte tethering or rolling / endothelial cell apoptotic process / necroptotic signaling pathway / positive regulation of synoviocyte proliferation / regulation of establishment of endothelial barrier / positive regulation of mononuclear cell migration / negative regulation of oxidative phosphorylation / macrophage activation involved in immune response / response to hydrogen sulfide / positive regulation of protein-containing complex disassembly / positive regulation of osteoclast differentiation / cellular response to toxic substance / positive regulation of macrophage derived foam cell differentiation / positive regulation of chemokine (C-X-C motif) ligand 2 production / regulation of fat cell differentiation / tumor necrosis factor receptor binding / positive regulation of heterotypic cell-cell adhesion / positive regulation of membrane protein ectodomain proteolysis / negative regulation of systemic arterial blood pressure / response to L-glutamate / positive regulation of extrinsic apoptotic signaling pathway / regulation of reactive oxygen species metabolic process / positive regulation of leukocyte adhesion to vascular endothelial cell / regulation of canonical NF-kappaB signal transduction / TNFR1-induced proapoptotic signaling / negative regulation of fat cell differentiation / positive regulation of cytokine production involved in inflammatory response / positive regulation of programmed cell death / TNFR1-mediated ceramide production / negative regulation of heart rate / negative regulation of viral genome replication / positive regulation of DNA biosynthetic process / positive regulation of amyloid-beta formation / positive regulation of neuroinflammatory response / negative regulation of endothelial cell proliferation / negative regulation of interleukin-6 production / positive regulation of immunoglobulin production / negative regulation of bicellular tight junction assembly / extrinsic apoptotic signaling pathway via death domain receptors / response to isolation stress / Interleukin-10 signaling / regulation of synapse organization / regulation of insulin secretion / histone H3K9ac reader activity / negative regulation of apoptotic signaling pathway / negative regulation of blood vessel endothelial cell migration / negative regulation of lipid storage / positive regulation of glial cell proliferation / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / detection of mechanical stimulus involved in sensory perception of pain / negative regulation of lipid catabolic process / extrinsic apoptotic signaling pathway / negative regulation of osteoblast differentiation / positive regulation of synaptic transmission / cellular response to retinoic acid / positive regulation of vascular associated smooth muscle cell proliferation / cell surface receptor signaling pathway via JAK-STAT / regulation of synaptic transmission, glutamatergic / positive regulation of chemokine production / phagocytic cup / skeletal muscle contraction
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 speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.43 Å
AuthorsTanaka, Y. / Sato, K. / Mima, M. / Mishima-Tsumagari, C.
Funding support Japan, 1items
OrganizationGrant numberCountry
Other private Japan
CitationJournal: Biochem Biophys Res Commun / Year: 2026
Title: Cryo-EM elucidates the interaction mechanism of ozoralizumab, a humanized anti-TNFα NANOBODY® compound.
Authors: Masashi Mima / Kyohei Sato / Takeshi Yokoyama / Chiemi Mishima-Tsumagari / Tatsuya Ohnuki / Yoshikazu Tanaka / Kunihiko Iwamoto /
Abstract: Ozoralizumab (OZR) is a next-generation TNF inhibitor composed of two identical humanized anti-TNFα NANOBODY® molecules (TNF30s) recombinantly linked via one humanized anti-human serum albumin (HSA) ...Ozoralizumab (OZR) is a next-generation TNF inhibitor composed of two identical humanized anti-TNFα NANOBODY® molecules (TNF30s) recombinantly linked via one humanized anti-human serum albumin (HSA) NANOBODY® molecule (ALB8) and two peptide linkers. OZR is designed as a unique format to exert potent inhibitory effects against TNFα with long plasma half-life. However, the three-dimensional structure of OZR-TNFα-HSA complex has not yet been elucidated, and a complete understanding of its interaction mechanism with TNFα is yet to be gained. In this study, we successfully observed the formation of the OZR-TNFα-HSA ternary complex by single-particle cryo-electron microscopy. The single-particle analysis revealed that the two TNF30 molecules of OZR simultaneously bind bivalently to TNFα in a 1:1-bivalent binding mode, while the ALB8 molecule binds to HSA, forming a ternary complex. Thus, OZR exhibits a binding mode significantly different from that of other IgG-type TNFα inhibitors. Furthermore, surface plasmon resonance (SPR) analysis demonstrated that the 1:1-bivalent binding mode confers an exceptionally slow dissociation rate, thereby contributing to the potent TNFα-neutralizing activity of OZR. These findings not only lend support to the favorable clinical efficacy of OZR from a structural standpoint but also lay the foundation for the rational design and development of next-generation TNFα inhibitors with enhanced and sustained efficacy.
History
DepositionDec 24, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 29, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data 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: anti-TNF-alpha nanobodies, TNF30, derived from Ozoralizumab (OZR)
B: Tumor necrosis factor
C: Tumor necrosis factor
D: Tumor necrosis factor


Theoretical massNumber of molelcules
Total (without water)90,7554
Polymers90,7554
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein anti-TNF-alpha nanobodies, TNF30, derived from Ozoralizumab (OZR)


Mass: 38471.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#2: Protein Tumor necrosis factor / Cachectin / TNF-alpha / Tumor necrosis factor ligand superfamily member 2 / TNF-a


Mass: 17427.709 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TNF, TNFA, TNFSF2 / Production host: Escherichia coli (E. coli) / References: UniProt: P01375
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

Component
IDNameTypeEntity IDParent-IDSource
1TNF-alpha in complex with the TNF-alpha inhibitor Ozoralizumab (OZR)COMPLEXall0RECOMBINANT
2Ozoralizumab (OZR)COMPLEX#11RECOMBINANT
3Tumor necrosis factor-alpha (TNF-alpha)COMPLEX#21RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Cricetulus griseus (Chinese hamster)10029
33Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21_5207model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 388293 / Symmetry type: POINT

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