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- PDB-6oge: Cryo-EM structure of Her2 extracellular domain-Trastuzumab Fab-Pe... -

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Basic information

Entry
Database: PDB / ID: 6oge
TitleCryo-EM structure of Her2 extracellular domain-Trastuzumab Fab-Pertuzumab Fab complex
Components
  • (Pertuzumab FAB ...) x 2
  • (Trastuzumab FAB ...) x 2
  • Receptor tyrosine-protein kinase erbB-2
Keywordstransferase/immune system / Her2 extracellular domain / Trastuzumab / Pertuzumab / transferase-immune system complex
Function / homology
Function and homology information


ERBB3:ERBB2 complex / RNA polymerase I core binding / negative regulation of immature T cell proliferation in thymus / positive regulation of transcription by RNA polymerase I / ErbB-3 class receptor binding / peripheral nervous system development / regulation of microtubule-based process / myelination / motor neuron axon guidance / immunoglobulin complex ...ERBB3:ERBB2 complex / RNA polymerase I core binding / negative regulation of immature T cell proliferation in thymus / positive regulation of transcription by RNA polymerase I / ErbB-3 class receptor binding / peripheral nervous system development / regulation of microtubule-based process / myelination / motor neuron axon guidance / immunoglobulin complex / neuromuscular junction development / oligodendrocyte differentiation / enzyme linked receptor protein signaling pathway / negative regulation of ERBB signaling pathway / positive regulation of cell adhesion / phosphatidylinositol 3-kinase signaling / regulation of cell motility / regulation of angiogenesis / positive regulation of protein targeting to membrane / complement activation / basal plasma membrane / immunoglobulin complex, circulating / immunoglobulin receptor binding / phagocytosis, recognition / positive regulation of B cell activation / cellular response to epidermal growth factor stimulus / regulation of ERK1 and ERK2 cascade / positive regulation of epithelial cell proliferation / neuron differentiation / positive regulation of translation / phagocytosis, engulfment / ERBB2 signaling pathway / antigen binding / complement activation, classical pathway / regulation of complement activation / cellular response to growth factor stimulus / positive regulation of GTPase activity / receptor protein-tyrosine kinase / transmembrane receptor protein tyrosine kinase activity / transmembrane signaling receptor activity / positive regulation of kinase activity / B cell receptor signaling pathway / peptidyl-tyrosine phosphorylation / positive regulation of MAP kinase activity / Fc-gamma receptor signaling pathway involved in phagocytosis / receptor-mediated endocytosis / wound healing / Fc-epsilon receptor signaling pathway / retina homeostasis / myelin sheath / transmembrane receptor protein tyrosine kinase signaling pathway / regulation of immune response / heart development / positive regulation of MAPK cascade / leukocyte migration / apical plasma membrane / positive regulation of cell growth / basolateral plasma membrane / protein tyrosine kinase activity / adaptive immune response / protein phosphatase binding / protein C-terminus binding / early endosome / blood microparticle / endosome membrane / receptor complex / cell surface receptor signaling pathway / MAPK cascade / intracellular signal transduction / immune response / protein heterodimerization activity / positive regulation of protein kinase B signaling / positive regulation of protein phosphorylation / protein autophosphorylation / external side of plasma membrane / regulation of transcription by RNA polymerase II / defense response to bacterium / multicellular organism development / innate immune response / protein phosphorylation / positive regulation of cell population proliferation / signal transduction / perinuclear region of cytoplasm / integral component of plasma membrane / extracellular space / extracellular exosome / integral component of membrane / extracellular region / ATP binding / identical protein binding / plasma membrane / nucleus / cytosol
Growth factor receptor domain IV / Protein kinase-like domain superfamily / Receptor L-domain / Protein kinase domain / Serine-threonine/tyrosine-protein kinase, catalytic domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin C1-set / Immunoglobulin subtype / Furin-like cysteine-rich domain / Furin-like repeat ...Growth factor receptor domain IV / Protein kinase-like domain superfamily / Receptor L-domain / Protein kinase domain / Serine-threonine/tyrosine-protein kinase, catalytic domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin C1-set / Immunoglobulin subtype / Furin-like cysteine-rich domain / Furin-like repeat / Immunoglobulin-like domain / Growth factor receptor cysteine-rich domain superfamily / Tyrosine-protein kinase, active site / Immunoglobulin V-set domain / Immunoglobulin-like domain superfamily / Receptor L domain / Furin-like cysteine rich region / Immunoglobulin-like fold / Receptor L-domain superfamily / Growth factor receptor domain 4 / Tyrosine-protein kinase, catalytic domain / Protein kinase, ATP binding site / Tyrosine protein kinase, EGF/ERB/XmrK receptor
Receptor tyrosine-protein kinase erbB-2 / Immunoglobulin gamma-1 heavy chain / IGH@ protein / Immunoglobulin kappa constant
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.36 Å
AuthorsHao, Y. / Yu, X. / Bai, Y. / Huang, X.
CitationJournal: PLoS ONE / Year: 2019
Title: Cryo-EM Structure of HER2-trastuzumab-pertuzumab complex.
Authors: Yue Hao / Xinchao Yu / Yonghong Bai / Helen J McBride / Xin Huang /
Abstract: Trastuzumab and pertuzumab are monoclonal antibodies that bind to distinct subdomains of the extracellular domain of human epidermal growth factor receptor 2 (HER2). Adding these monoclonal ...Trastuzumab and pertuzumab are monoclonal antibodies that bind to distinct subdomains of the extracellular domain of human epidermal growth factor receptor 2 (HER2). Adding these monoclonal antibodies to the treatment regimen of HER2-positive breast cancer has changed the paradigm for treatment in that form of cancer. Synergistic activity has been observed with the combination of these two antibodies leading to hypotheses regarding the mechanism(s) and to the development of bispecific antibodies to maximize the clinical effect further. Although the individual crystal structures of HER2-trastuzumab and HER2-pertuzumab revealed the distinct binding sites and provided the structural basis for their anti-tumor activities, detailed structural information on the HER2-trastuzumab-pertuzumab complex has been elusive. Here we present the cryo-EM structure of HER2-trastuzumab-pertuzumab at 4.36 Å resolution. Comparison with the binary complexes reveals no cooperative interaction between trastuzumab and pertuzumab, and provides key insights into the design of novel, high-avidity bispecific molecules with potentially greater clinical efficacy.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 2, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 15, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
A: Receptor tyrosine-protein kinase erbB-2
B: Pertuzumab FAB LIGHT CHAIN
C: Pertuzumab FAB HEAVY CHAIN
D: Trastuzumab FAB LIGHT CHAIN
E: Trastuzumab FAB HEAVY CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,55914
Polymers162,6515
Non-polymers1,9099
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Receptor tyrosine-protein kinase erbB-2 / Metastatic lymph node gene 19 protein / MLN 19 / Proto-oncogene Neu / Proto-oncogene c-ErbB-2 / ...Metastatic lymph node gene 19 protein / MLN 19 / Proto-oncogene Neu / Proto-oncogene c-ErbB-2 / Tyrosine kinase-type cell surface receptor HER2 / p185erbB2


Mass: 68536.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ERBB2, HER2, MLN19, NEU, NGL / Production host: Homo sapiens (human)
References: UniProt: P04626, receptor protein-tyrosine kinase

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Antibody , 4 types, 4 molecules BCDE

#2: Antibody Pertuzumab FAB LIGHT CHAIN


Mass: 23548.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IGKC / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P01834
#3: Antibody Pertuzumab FAB HEAVY CHAIN


Mass: 23674.486 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) / References: UniProt: P0DOX5
#4: Antibody Trastuzumab FAB LIGHT CHAIN


Mass: 23466.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IGKC / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P01834
#5: Antibody Trastuzumab FAB HEAVY CHAIN


Mass: 23425.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IGH@ / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: Q6GMX6

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Sugars , 3 types, 9 molecules

#6: Sugar
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Mass: 221.208 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
#7: Sugar ChemComp-BMA / BETA-D-MANNOSE / Mannose


Mass: 180.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
#8: Sugar ChemComp-MAN / ALPHA-D-MANNOSE / Mannose


Mass: 180.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H12O6

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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
1Her2 extracellular domain-Trastuzumab Fab-Pertuzumab Fab complexCOMPLEX1, 2, 3, 4, 50MULTIPLE SOURCES
2Human HER2 extracellular domainCOMPLEX11RECOMBINANT
3Pertuzumab FabCOMPLEX2, 31RECOMBINANT
4Trastuzumab FabCOMPLEX4, 51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Cricetulus griseus (Chinese hamster)10029
44Cricetulus griseus (Chinese hamster)10029
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 2.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: -1500 nm / Nominal defocus min: -3500 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 6 sec. / Electron dose: 45 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.13rc2_2986: / Classification: refinement
EM software
IDNameCategory
1cisTEMparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
9cisTEMinitial Euler assignment
12cisTEM3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1032611
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 398409 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00811809
ELECTRON MICROSCOPYf_angle_d1.17416110
ELECTRON MICROSCOPYf_dihedral_angle_d7.0817014
ELECTRON MICROSCOPYf_chiral_restr0.0571810
ELECTRON MICROSCOPYf_plane_restr0.0092080

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