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- EMDB-67993: Cryo-EM structure of the TNF-alpha-Ozoralizumab (OZR)-HSA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-67993
TitleCryo-EM structure of the TNF-alpha-Ozoralizumab (OZR)-HSA complex
Map data
Sample
  • Complex: TNF-alpha-Ozoralizumab (OZR)-HSA complex
    • Complex: Ozoralizumab (OZR)
      • Protein or peptide: Ozoralizumab (OZR)
    • Complex: Tumor necrosis factor-alpha (TNF-alpha)
      • Protein or peptide: Tumor necrosis factor
    • Complex: Human Serum Albumin (HSA)
      • Protein or peptide: Serum albumin
KeywordsNANOBODY / VHH / TNF-alpha / HSA / Ozoralizumab / OZR / CYTOKINE
Function / homology
Function and homology information


response to Gram-negative bacterium / negative regulation of L-glutamate import across plasma membrane / positive regulation of chronic inflammatory response to antigenic stimulus / 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 Gram-negative bacterium / negative regulation of L-glutamate import across plasma membrane / positive regulation of chronic inflammatory response to antigenic stimulus / 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 / death receptor agonist activity / positive regulation of translational initiation by iron / positive regulation of protein transport / positive regulation of vitamin D biosynthetic process / regulation of branching involved in salivary gland morphogenesis / regulation of endothelial cell apoptotic process / chronic inflammatory response to antigenic stimulus / negative regulation of protein-containing complex disassembly / response to macrophage colony-stimulating factor / positive regulation of humoral immune response mediated by circulating immunoglobulin / positive regulation of leukocyte adhesion to arterial endothelial cell / response to gold nanoparticle / negative regulation of myelination / positive regulation of JUN kinase activity / negative regulation of vascular wound healing / negative regulation of amyloid-beta clearance / negative regulation of cytokine production involved in immune response / reactive gliosis / positive regulation of hair follicle development / positive regulation of interleukin-18 production / inflammatory response to wounding / response to resveratrol / epithelial cell proliferation involved in salivary gland morphogenesis / positive regulation of action potential / response to quercetin / TNF signaling / toll-like receptor 3 signaling pathway / negative regulation of D-glucose import across plasma membrane / vascular endothelial growth factor production / embryonic digestive tract development / positive regulation of fever generation / positive regulation of calcineurin-NFAT signaling cascade / necroptotic signaling pathway / positive regulation of synoviocyte proliferation / leukocyte tethering or rolling / negative regulation of myoblast differentiation / bilirubin transport / positive regulation of mononuclear cell migration / response to fructose / positive regulation of hepatocyte proliferation / regulation of establishment of endothelial barrier / endothelial cell apoptotic process / negative regulation of oxidative phosphorylation / response to hydrogen sulfide / Ciprofloxacin ADME / positive regulation of protein-containing complex disassembly / exogenous protein binding / cellular response to calcium ion starvation / positive regulation of protein localization to cell surface / positive regulation of osteoclast differentiation / cellular response to toxic substance / macrophage activation involved in immune response / positive regulation of macrophage derived foam cell differentiation / tumor necrosis factor receptor binding / negative regulation of mitotic cell cycle / positive regulation of chemokine (C-X-C motif) ligand 2 production / positive regulation of cytokine production involved in inflammatory response / enterobactin binding / positive regulation of podosome assembly / regulation of fat cell differentiation / Heme biosynthesis / positive regulation of heterotypic cell-cell adhesion / positive regulation of programmed cell death / positive regulation of membrane protein ectodomain proteolysis / regulation of canonical NF-kappaB signal transduction / HDL remodeling / molecular carrier activity / response to L-glutamate / negative regulation of mitochondrial depolarization / positive regulation of leukocyte adhesion to vascular endothelial cell / negative regulation of systemic arterial blood pressure / TNFR1-induced proapoptotic signaling / positive regulation of extrinsic apoptotic signaling pathway / Prednisone ADME / TNFR1-mediated ceramide production / Heme degradation / mRNA stabilization / regulation of reactive oxygen species metabolic process / negative regulation of heart rate / positive regulation of DNA biosynthetic process / positive regulation of amyloid-beta formation / positive regulation of neuroinflammatory response / negative regulation of viral genome replication / positive regulation of immunoglobulin production / negative regulation of bicellular tight junction assembly / negative regulation of fat cell differentiation / Aspirin ADME / negative regulation of endothelial cell proliferation / antioxidant activity
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 / Serum albumin/Alpha-fetoprotein/Afamin ...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 / Serum albumin/Alpha-fetoprotein/Afamin / ALB/AFP/VDB / Serum albumin, N-terminal / Serum albumin, conserved site / Serum albumin-like / Serum albumin family / Albumin domain signature. / Albumin domain profile. / serum albumin
Similarity search - Domain/homology
Tumor necrosis factor / Albumin
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.19 Å
AuthorsTanaka Y / Sato K / Mima M / Mishima-Tsumagari C
Funding support Japan, 1 items
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 26, 2025-
Header (metadata) releaseApr 29, 2026-
Map releaseApr 29, 2026-
UpdateApr 29, 2026-
Current statusApr 29, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_67993.png

Downloads & links

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Map

FileDownload / File: emd_67993.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.79 Å/pix.
x 384 pix.
= 302.592 Å		0.79 Å/pix.
x 384 pix.
= 302.592 Å		0.79 Å/pix.
x 384 pix.
= 302.592 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.788 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.030968048 - 0.07898633
Average (Standard dev.)0.000014571411 (±0.0023090038)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 302.59198 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_67993_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_67993_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : TNF-alpha-Ozoralizumab (OZR)-HSA complex

EntireName: TNF-alpha-Ozoralizumab (OZR)-HSA complex
Components
  • Complex: TNF-alpha-Ozoralizumab (OZR)-HSA complex
    • Complex: Ozoralizumab (OZR)
      • Protein or peptide: Ozoralizumab (OZR)
    • Complex: Tumor necrosis factor-alpha (TNF-alpha)
      • Protein or peptide: Tumor necrosis factor
    • Complex: Human Serum Albumin (HSA)
      • Protein or peptide: Serum albumin

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Supramolecule #1: TNF-alpha-Ozoralizumab (OZR)-HSA complex

SupramoleculeName: TNF-alpha-Ozoralizumab (OZR)-HSA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Ozoralizumab (OZR)

SupramoleculeName: Ozoralizumab (OZR) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Tumor necrosis factor-alpha (TNF-alpha)

SupramoleculeName: Tumor necrosis factor-alpha (TNF-alpha) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Human Serum Albumin (HSA)

SupramoleculeName: Human Serum Albumin (HSA) / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ozoralizumab (OZR)

MacromoleculeName: Ozoralizumab (OZR) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 38.471664 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMYWVRQA PGKGLEWVSE INTNGLITKY PDSVKGRFTI SRDNAKNTLY LQMNSLRPE DTAVYYCARS PSGFNRGQGT LVTVSSGGGG SGGGSEVQLV ESGGGLVQPG NSLRLSCAAS GFTFSSFGMS W VRQAPGKG ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMYWVRQA PGKGLEWVSE INTNGLITKY PDSVKGRFTI SRDNAKNTLY LQMNSLRPE DTAVYYCARS PSGFNRGQGT LVTVSSGGGG SGGGSEVQLV ESGGGLVQPG NSLRLSCAAS GFTFSSFGMS W VRQAPGKG LEWVSSISGS GSDTLYADSV KGRFTISRDN AKTTLYLQMN SLRPEDTAVY YCTIGGSLSR SSQGTLVTVS SG GGGSGGG SEVQLVESGG GLVQPGGSLR LSCAASGFTF SDYWMYWVRQ APGKGLEWVS EINTNGLITK YPDSVKGRFT ISR DNAKNT LYLQMNSLRP EDTAVYYCAR SPSGFNRGQG TLVTVSS

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Macromolecule #2: Tumor necrosis factor

MacromoleculeName: Tumor necrosis factor / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.427709 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GVRSSSRTPS DKPVAHVVAN PQAEGQLQWL NRRANALLAN GVELRDNQLV VPSEGLYLIY SQVLFKGQGC PSTHVLLTHT ISRIAVSYQ TKVNLLSAIK SPCQRETPEG AEAKPWYEPI YLGGVFQLEK GDRLSAEINR PDYLDFAESG QVYFGIIAL

UniProtKB: Tumor necrosis factor

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Macromolecule #3: Serum albumin

MacromoleculeName: Serum albumin / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 66.571219 KDa
SequenceString: DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMAD CCAKQEPERN ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF YAPELLFFAK R YKAAFTEC ...String:
DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMAD CCAKQEPERN ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF YAPELLFFAK R YKAAFTEC CQAADKAACL LPKLDELRDE GKASSAKQRL KCASLQKFGE RAFKAWAVAR LSQRFPKAEF AEVSKLVTDL TK VHTECCH GDLLECADDR ADLAKYICEN QDSISSKLKE CCEKPLLEKS HCIAEVENDE MPADLPSLAA DFVESKDVCK NYA EAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGE YKFQN ALLVRYTKKV PQVSTPTLVE VSRNLGKVGS KCCKHPEAKR MPCAEDYLSV VLNQLCVLHE KTPVSDRVTK CCTES LVNR RPCFSALEVD ETYVPKEFNA ETFTFHADIC TLSEKERQIK KQTALVELVK HKPKATKEQL KAVMDDFAAF VEKCCK ADD KETCFAEEGK KLVAASQAAL GL

UniProtKB: Albumin

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.7000000000000001 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8z8v


Details: 8Z8M
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.19 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 41373
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

8z8v


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-21tv:
Cryo-EM structure of the TNF-alpha-Ozoralizumab (OZR)-HSA complex

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