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- EMDB-60764: Local refinement structure of sEGFR and 528 Fv (from HL-type bisp... -

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Entry
Database: EMDB / ID: EMD-60764
TitleLocal refinement structure of sEGFR and 528 Fv (from HL-type bispecific diabody Ex3) complex
Map data
Sample
  • Complex: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
    • Complex: Soluble Epidermal Growth Factor Receptor (sEGFR)
      • Protein or peptide: Epidermal growth factor receptor
    • Complex: HL-type bispecific diabody Ex3
      • Protein or peptide: 528 Fv from HL-type bispecific diabody Ex3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsbispecific antibody / diabody / EGFR / HL / Ex3 / 528 / local refinement / ANTITUMOR PROTEIN / ANTITUMOR PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


positive regulation of protein kinase C activity / multivesicular body, internal vesicle lumen / negative regulation of cardiocyte differentiation / response to hydroxyisoflavone / diterpenoid metabolic process / positive regulation of prolactin secretion / Shc-EGFR complex / Inhibition of Signaling by Overexpressed EGFR / ovulation cycle / EGFR interacts with phospholipase C-gamma ...positive regulation of protein kinase C activity / multivesicular body, internal vesicle lumen / negative regulation of cardiocyte differentiation / response to hydroxyisoflavone / diterpenoid metabolic process / positive regulation of prolactin secretion / Shc-EGFR complex / Inhibition of Signaling by Overexpressed EGFR / ovulation cycle / EGFR interacts with phospholipase C-gamma / positive regulation of mucus secretion / regulation of peptidyl-tyrosine phosphorylation / epidermal growth factor binding / tongue development / response to UV-A / PLCG1 events in ERBB2 signaling / midgut development / ERBB2-EGFR signaling pathway / hydrogen peroxide metabolic process / morphogenesis of an epithelial fold / PTK6 promotes HIF1A stabilization / ERBB2 Activates PTK6 Signaling / digestive tract morphogenesis / Signaling by EGFR / response to cobalamin / intracellular vesicle / eyelid development in camera-type eye / negative regulation of epidermal growth factor receptor signaling pathway / cerebral cortex cell migration / protein insertion into membrane / ERBB2 Regulates Cell Motility / hepatocyte growth factor receptor activity / platelet-derived growth factor beta-receptor activity / placental growth factor receptor activity / brain-derived neurotrophic factor receptor activity / boss receptor activity / platelet-derived growth factor alpha-receptor activity / epidermal growth factor receptor activity / macrophage colony-stimulating factor receptor activity / protein tyrosine kinase activator activity / protein tyrosine kinase collagen receptor activity / stem cell factor receptor activity / Respiratory syncytial virus (RSV) attachment and entry / positive regulation of bone resorption / Signaling by ERBB4 / PI3K events in ERBB2 signaling / negative regulation of mitotic cell cycle / insulin-like growth factor receptor activity / transmembrane-ephrin receptor activity / positive regulation of phosphorylation / GPI-linked ephrin receptor activity / positive regulation of peptidyl-serine phosphorylation / vascular endothelial growth factor receptor activity / peptidyl-tyrosine autophosphorylation / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / hair follicle development / MAP kinase kinase kinase activity / GAB1 signalosome / positive regulation of G1/S transition of mitotic cell cycle / fibroblast growth factor receptor activity / embryonic placenta development / insulin receptor activity / salivary gland morphogenesis / positive regulation of vasoconstriction / neuron projection morphogenesis / Signaling by ERBB2 / positive regulation of glial cell proliferation / GRB2 events in EGFR signaling / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors / SHC1 events in EGFR signaling / transmembrane receptor protein tyrosine kinase activity / EGFR Transactivation by Gastrin / cellular response to dexamethasone stimulus / GRB2 events in ERBB2 signaling / positive regulation of synaptic transmission, glutamatergic / SHC1 events in ERBB2 signaling / ossification / NOTCH3 Activation and Transmission of Signal to the Nucleus / positive regulation of DNA repair / basal plasma membrane / cellular response to epidermal growth factor stimulus / positive regulation of epithelial cell proliferation / EGFR downregulation / positive regulation of superoxide anion generation / positive regulation of DNA replication / epithelial cell proliferation / Signal transduction by L1 / positive regulation of smooth muscle cell proliferation / positive regulation of protein localization to plasma membrane / liver regeneration / astrocyte activation / cellular response to amino acid stimulus / cellular response to estradiol stimulus / phosphatidylinositol 3-kinase/protein kinase B signal transduction / lung development / synaptic membrane / peptidyl-tyrosine phosphorylation / clathrin-coated endocytic vesicle membrane / Signaling by ERBB2 TMD/JMD mutants / Constitutive Signaling by EGFRvIII
Similarity search - Function
: / Epidermal growth factor receptor transmembrane-juxtamembrane segment / Tyrosine protein kinase, EGF/ERB/XmrK receptor / Growth factor receptor domain 4 / Growth factor receptor domain IV / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain ...: / Epidermal growth factor receptor transmembrane-juxtamembrane segment / Tyrosine protein kinase, EGF/ERB/XmrK receptor / Growth factor receptor domain 4 / Growth factor receptor domain IV / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain / Furin-like repeat / Furin-like repeats / Growth factor receptor cysteine-rich domain superfamily / : / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Epidermal growth factor receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsSato K / Uehara S / Tsugita A / Matsui T / Asano R / Makabe K / Yokoyama T / Tanaka Y
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Cell Rep / Year: 2025
Title: Bispecific antibody-antigen complex structures reveal activity enhancement by domain rearrangement.
Authors: Kyohei Sato / Shiro Uehara / Atsushi Tsugita / Mayuka Ishii / Shieru Ishiyama / Atsushi Maejima / Ishin Nakahara / Misae Nazuka / Takashi Matsui / Gatsogiannis Christos / Takeshi Yokoyama / ...Authors: Kyohei Sato / Shiro Uehara / Atsushi Tsugita / Mayuka Ishii / Shieru Ishiyama / Atsushi Maejima / Ishin Nakahara / Misae Nazuka / Takashi Matsui / Gatsogiannis Christos / Takeshi Yokoyama / Izumi Kumagai / Koki Makabe / Ryutaro Asano / Yoshikazu Tanaka /
Abstract: Bispecific antibodies (BsAbs) have been developed as anti-cancer drugs that accumulate activated T cells on cancer cells by bridging the antigens present in each cell. Ex3 is a diabody-type BsAb ...Bispecific antibodies (BsAbs) have been developed as anti-cancer drugs that accumulate activated T cells on cancer cells by bridging the antigens present in each cell. Ex3 is a diabody-type BsAb composed of an anti-epidermal growth factor receptor (EGFR) antibody and an anti-CD3 antibody. In the design of Ex3, the LH-type domain order (Ex3LH) is shown to have more than 100-fold greater anti-cancer activity than the HL-type domain order (Ex3HL). To understand this phenomenon of activity enhancement by domain-order rearrangement, we report here cryoelectron microscopy (cryo-EM) structures of both Ex3HL and Ex3LH in complex with EGFR and CD3. A structural comparison of the HL and LH types reveals that the domain rearrangement leads to drastic structural changes and that the avoidance of steric hindrance by a favorable bridging angle on the cell surface is the fundamental mechanism for this activity enhancement.
History
DepositionJul 10, 2024-
Header (metadata) releaseMay 28, 2025-
Map releaseMay 28, 2025-
UpdateAug 6, 2025-
Current statusAug 6, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60764.png

Downloads & links

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Map

FileDownload / File: emd_60764.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: 0.065
Minimum - Maximum-0.29388818 - 0.48055947
Average (Standard dev.)0.00009781776 (±0.0045876047)
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_60764_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_60764_half_map_2.map
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Histogram (log scale)

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Sample components

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Entire : HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in t...

EntireName: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
Components
  • Complex: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
    • Complex: Soluble Epidermal Growth Factor Receptor (sEGFR)
      • Protein or peptide: Epidermal growth factor receptor
    • Complex: HL-type bispecific diabody Ex3
      • Protein or peptide: 528 Fv from HL-type bispecific diabody Ex3
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in t...

SupramoleculeName: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Soluble Epidermal Growth Factor Receptor (sEGFR)

SupramoleculeName: Soluble Epidermal Growth Factor Receptor (sEGFR) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: HL-type bispecific diabody Ex3

SupramoleculeName: HL-type bispecific diabody Ex3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Epidermal growth factor receptor

MacromoleculeName: Epidermal growth factor receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.496062 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: LEEKKVCQGT SNKLTQLGTF EDHFLSLQRM FNNCEVVLGN LEITYVQRNY DLSFLKTIQE VAGYVLIALN TVERIPLENL QIIRGNMYY ENSYALAVLS NYDANKTGLK ELPMRNLQEI LHGAVRFSNN PALCNVESIQ WRDIVSSDFL SNMSMDFQNH L GSCQKCDP ...String:
LEEKKVCQGT SNKLTQLGTF EDHFLSLQRM FNNCEVVLGN LEITYVQRNY DLSFLKTIQE VAGYVLIALN TVERIPLENL QIIRGNMYY ENSYALAVLS NYDANKTGLK ELPMRNLQEI LHGAVRFSNN PALCNVESIQ WRDIVSSDFL SNMSMDFQNH L GSCQKCDP SCPNGSCWGA GEENCQKLTK IICAQQCSGR CRGKSPSDCC HNQCAAGCTG PRESDCLVCR KFRDEATCKD TC PPLMLYN PTTYQMDVNP EGKYSFGATC VKKCPRNYVV TDHGSCVRAC GADSYEMEED GVRKCKKCEG PCRKVCNGIG IGE FKDSLS INATNIKHFK NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF ENLE IIRGR TKQHGQFSLA VVSLNITSLG LRSLKEISDG DVIISGNKNL CYANTINWKK LFGTSGQKTK IISNRGENSC KATGQ VCHA LCSPEGCWGP EPRDCVSCRN VSRGRECVDK CNLLEGEPRE FVENSECIQC HPECLPQAMN ITCTGRGPDN CIQCAH YID GPHCVKTCPA GVMGENNTLV WKYADAGHVC HLCHPNCTYG CTGPGLEGCP TNGPKIPSHH HHHH

UniProtKB: Epidermal growth factor receptor

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Macromolecule #2: 528 Fv from HL-type bispecific diabody Ex3

MacromoleculeName: 528 Fv from HL-type bispecific diabody Ex3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 27.093129 KDa
Recombinant expressionOrganism: Brevibacillus choshinensis (bacteria)
SequenceString: DIVMTQSPLS LPVTPGEPAS ISCRSSQNIV HNNGITYLEW YLQKPGQSPQ LLIYKVSDRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVG VYYCFQGSHI PPTFGQGTKV EIKRAAAAGG GGSGGGGSGG GGSGGGGSQV QLVQSGAEVK KPGASVKVSC K ASGYTFTS ...String:
DIVMTQSPLS LPVTPGEPAS ISCRSSQNIV HNNGITYLEW YLQKPGQSPQ LLIYKVSDRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVG VYYCFQGSHI PPTFGQGTKV EIKRAAAAGG GGSGGGGSGG GGSGGGGSQV QLVQSGAEVK KPGASVKVSC K ASGYTFTS YWMHWVRQAP GQGLEWMGNI WPGSGGTNYA EKFKNRVTMT RDTSISTAYM ELSRLRSDDT AVYYCARSGG PY FFDYWGQ GTLVTVSS

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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: 7.4
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: 60.0 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: 0.9 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 781005
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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