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- EMDB-63050: Cryo-EM structure of linker-extended biparatopic antibody BA1-GP4... -

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Basic information

Entry
Database: EMDB / ID: EMD-63050
TitleCryo-EM structure of linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2
Map data
Sample
  • Complex: Linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2
    • Protein or peptide: Tumor necrosis factor receptor superfamily member 1B
    • Protein or peptide: TR92 heavy chain
    • Protein or peptide: TR92 light chain
    • Protein or peptide: TR96 heavy chain
    • Protein or peptide: TR96 light chain
KeywordsAntibody / biparatopic antibody / antagonist / IMMUNE SYSTEM
Function / homology
Function and homology information


glial cell-neuron signaling / regulation of cytokine production involved in immune response / tumor necrosis factor receptor superfamily complex / pulmonary valve development / RNA destabilization / aortic valve development / tumor necrosis factor receptor activity / negative regulation of extracellular matrix constituent secretion / positive regulation of apoptotic process involved in morphogenesis / regulation of T cell cytokine production ...glial cell-neuron signaling / regulation of cytokine production involved in immune response / tumor necrosis factor receptor superfamily complex / pulmonary valve development / RNA destabilization / aortic valve development / tumor necrosis factor receptor activity / negative regulation of extracellular matrix constituent secretion / positive regulation of apoptotic process involved in morphogenesis / regulation of T cell cytokine production / negative regulation of neuroinflammatory response / TNFs bind their physiological receptors / tumor necrosis factor binding / negative regulation of cardiac muscle hypertrophy / regulation of neuroinflammatory response / positive regulation of myelination / positive regulation of membrane protein ectodomain proteolysis / regulation of myelination / Interleukin-10 signaling / regulation of T cell proliferation / positive regulation of oligodendrocyte differentiation / specific granule membrane / extrinsic apoptotic signaling pathway / TNFR2 non-canonical NF-kB pathway / tumor necrosis factor-mediated signaling pathway / intrinsic apoptotic signaling pathway in response to DNA damage / cellular response to lipopolysaccharide / Interleukin-4 and Interleukin-13 signaling / inflammatory response / membrane raft / ubiquitin protein ligase binding / Neutrophil degranulation / extracellular region / membrane / plasma membrane
Similarity search - Function
Tumour necrosis factor receptor 1B / Tumor necrosis factor receptor 1B, N-terminal / : / TNFR/NGFR family cysteine-rich region domain profile. / TNFR/NGFR cysteine-rich region / TNFR/NGFR family cysteine-rich region signature. / Tumor necrosis factor receptor / nerve growth factor receptor repeats. / TNFR/NGFR cysteine-rich region
Similarity search - Domain/homology
Tumor necrosis factor receptor superfamily member 1B
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsOtsuki T / Matsumoto S / Fujita J / Miyata T / Namba K / Kanada R / Okuno Y / Kamada H / Ohno H / Akiba H
Funding support Japan, 4 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP22ak0101099 Japan
Japan Agency for Medical Research and Development (AMED)JP24ama121042 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121003 Japan
Japan Society for the Promotion of Science (JSPS)JP24K01270 Japan
CitationJournal: J Biol Chem / Year: 2025
Title: Conversion of an agonistic anti-TNFR2 biparatopic antibody into an antagonist by insertion of peptide linkers into the hinge region.
Authors: Takuya Otsuki / Shigeyuki Matsumoto / Junso Fujita / Tomoko Miyata / Keiichi Namba / Ryo Kanada / Yasushi Okuno / Haruhiko Kamada / Hiroaki Ohno / Hiroki Akiba /
Abstract: Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or ...Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or cell internalization, whereas few were reported with smaller immunocomplexes to suppress unwanted signaling. Here, we developed a strategy to induce 1:1 immunocomplex formation to maximize antagonistic function. Various peptide linkers were introduced into the hinge regions of IgG-like agonist BpAbs against tumor necrosis factor receptor 2. Loss of crosslinking activity was observed for one BpAb, allowing the conversion of its function from an agonist to an antagonist. However, cross-linking activity was retained for another agonist BpAb, which binds to a different epitope pair. In a combined analysis of cryo-electron microscopy and coarse-grained molecular dynamics simulations, effect of epitope combination on the stability of 1:1 complexes was observed. These results lead to an understanding of the mechanism and design of BpAbs to adopt a 1:1-binding mode.
History
DepositionJan 8, 2025-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateAug 13, 2025-
Current statusAug 13, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_63050.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 300 pix.
= 314.4 Å
1.05 Å/pix.
x 300 pix.
= 314.4 Å
1.05 Å/pix.
x 300 pix.
= 314.4 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density
Contour LevelBy AUTHOR: 0.066
Minimum - Maximum-0.28885934 - 0.3818899
Average (Standard dev.)-0.0003448568 (±0.007968252)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 314.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_63050_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

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Half map: #2

Fileemd_63050_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_63050_half_map_2.map
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Sample components

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Entire : Linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2

EntireName: Linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2
Components
  • Complex: Linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2
    • Protein or peptide: Tumor necrosis factor receptor superfamily member 1B
    • Protein or peptide: TR92 heavy chain
    • Protein or peptide: TR92 light chain
    • Protein or peptide: TR96 heavy chain
    • Protein or peptide: TR96 light chain

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Supramolecule #1: Linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2

SupramoleculeName: Linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Tumor necrosis factor receptor superfamily member 1B

MacromoleculeName: Tumor necrosis factor receptor superfamily member 1B / type: protein_or_peptide / ID: 1
Details: 1-178, Tumor necrosis factor receptor 1B, Homo sapiens; 179-543, Maltose/maltodextrin-binding periplasmic protein - Escherichia coli K-12; 545-550, hexahistidine tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.747895 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
TPYAPEPGST CRLREYYDQT AQMCCSKCSP GQHAKVFCTK TSDTVCDSCE DSTYTQLWNW VPECLSCGSR CSSDQVETQA CTREQNRIC TCRPGWYCAL SKQEGCRLCA PLRKCRPGFG VARPGTETSD VVCKPCAPGT FSNTTSSTDI CRP

UniProtKB: Tumor necrosis factor receptor superfamily member 1B

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Macromolecule #2: TR92 heavy chain

MacromoleculeName: TR92 heavy chain / type: protein_or_peptide / ID: 2 / Details: TR92 antibody heavy chain / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.777564 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: KVQLQQSGAE LVKPGASVKL SCKASGYTFT ESIIHWVKQR SGQGLEWIGW FYPGSDNINY NEKFKDKATL TADKSSSTVY MELTRLTSE DSAVYFCASH EGPYVYFDYW GQGTTLTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS W NSGALTSG ...String:
KVQLQQSGAE LVKPGASVKL SCKASGYTFT ESIIHWVKQR SGQGLEWIGW FYPGSDNINY NEKFKDKATL TADKSSSTVY MELTRLTSE DSAVYFCASH EGPYVYFDYW GQGTTLTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS W NSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKS

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Macromolecule #3: TR92 light chain

MacromoleculeName: TR92 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.242812 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: IVMTQSHKFM STSVGDRVSI TCKASQDVST AVAWYQQKPG QSPKLLIYWT STRHTGVPDR FTGSGSGTDY TLTISSVQAE DLALYYCQH HYSTPYTFGG GTKLEIQRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTEQDSK ...String:
IVMTQSHKFM STSVGDRVSI TCKASQDVST AVAWYQQKPG QSPKLLIYWT STRHTGVPDR FTGSGSGTDY TLTISSVQAE DLALYYCQH HYSTPYTFGG GTKLEIQRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRG

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Macromolecule #4: TR96 heavy chain

MacromoleculeName: TR96 heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.093518 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLQQSGAE LVKPGASVKL SCTPSGFNIK DTYMHWVKQR PEQGLEWIGR IDPANGYTEY DPKFQDKATI TADTSSNTAY LQLSSLTSE DTAVYYCADT QLYYWGQGTT LTVSSASVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN A LQSGNSQE ...String:
EVQLQQSGAE LVKPGASVKL SCTPSGFNIK DTYMHWVKQR PEQGLEWIGR IDPANGYTEY DPKFQDKATI TADTSSNTAY LQLSSLTSE DTAVYYCADT QLYYWGQGTT LTVSSASVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN A LQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR G

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Macromolecule #5: TR96 light chain

MacromoleculeName: TR96 light chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 22.055656 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QIVLTQSPAI MSASLGERVT MTCTASSSVS STYLHWYQQK PGSSPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISNME AEDAATYYC HQYHRSPLTF GAGTKLELKS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG V HTFPAVLQ ...String:
QIVLTQSPAI MSASLGERVT MTCTASSSVS STYLHWYQQK PGSSPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISNME AEDAATYYC HQYHRSPLTF GAGTKLELKS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG V HTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EP

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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

Concentration0.71 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4098 pixel / Number grids imaged: 1 / Number real images: 5508 / Average exposure time: 4.87 sec. / Average electron dose: 80.0 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: 0.5 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 2971529
CTF correctionSoftware - Name: cryoSPARC (ver. 4.2.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 178242
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9lfl:
Cryo-EM structure of linker-extended biparatopic antibody BA1-GP4 in complex with TNFR2

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