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

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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


Classical antibody-mediated complement activation / Role of phospholipids in phagocytosis / Regulation of actin dynamics for phagocytic cup formation / FCGR activation / Cell surface interactions at the vascular wall / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / PI3K events in ERBB2 signaling / GRB2 events in ERBB2 signaling / Constitutive Signaling by Aberrant PI3K in Cancer / Initial triggering of complement ...Classical antibody-mediated complement activation / Role of phospholipids in phagocytosis / Regulation of actin dynamics for phagocytic cup formation / FCGR activation / Cell surface interactions at the vascular wall / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / PI3K events in ERBB2 signaling / GRB2 events in ERBB2 signaling / Constitutive Signaling by Aberrant PI3K in Cancer / Initial triggering of complement / Downregulation of ERBB2:ERBB3 signaling / GRB7 events in ERBB2 signaling / PIP3 activates AKT signaling / PLCG1 events in ERBB2 signaling / SHC1 events in ERBB2 signaling / Scavenging of heme from plasma / Signaling by ERBB2 / CD22 mediated BCR regulation / ERBB2 Regulates Cell Motility / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / ERBB2 Activates PTK6 Signaling / Downregulation of ERBB2 signaling / Sema4D induced cell migration and growth-cone collapse / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors / Regulation of Complement cascade / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / RAF/MAP kinase cascade / FCERI mediated NF-kB activation / FCERI mediated Ca+2 mobilization / FCERI mediated MAPK activation / Role of LAT2/NTAL/LAB on calcium mobilization / Fc epsilon receptor (FCERI) signaling / negative regulation of immature T cell proliferation in thymus / RNA polymerase I core binding / regulation of microtubule-based process / positive regulation of transcription by RNA polymerase I / ErbB-3 class receptor binding / peripheral nervous system development / oligodendrocyte differentiation / motor neuron axon guidance / myelination / neuromuscular junction development / enzyme linked receptor protein signaling pathway / negative regulation of ERBB signaling pathway / positive regulation of cell adhesion / phosphatidylinositol 3-kinase signaling / regulation of angiogenesis / regulation of cell motility / positive regulation of protein targeting to membrane / basal plasma membrane / complement activation / cellular response to growth factor stimulus / regulation of ERK1 and ERK2 cascade / immunoglobulin complex, circulating / immunoglobulin receptor binding / phagocytosis, recognition / positive regulation of B cell activation / cellular response to epidermal growth factor stimulus / positive regulation of translation / positive regulation of epithelial cell proliferation / ERBB2 signaling pathway / neuron differentiation / phagocytosis, engulfment / positive regulation of GTPase activity / complement activation, classical pathway / regulation of complement activation / antigen binding / receptor protein-tyrosine kinase / transmembrane receptor protein tyrosine kinase activity / Ras guanyl-nucleotide exchange factor activity / transmembrane signaling receptor activity / Fc-epsilon receptor signaling pathway / receptor-mediated endocytosis / positive regulation of MAP kinase activity / retina homeostasis / B cell receptor signaling pathway / wound healing / phosphatidylinositol-4,5-bisphosphate 3-kinase activity / Fc-gamma receptor signaling pathway involved in phagocytosis / leukocyte migration / transmembrane receptor protein tyrosine kinase signaling pathway / adaptive immune response / heart development / apical plasma membrane / regulation of immune response / positive regulation of MAPK cascade / myelin sheath / positive regulation of cell growth / basolateral plasma membrane / protein phosphatase binding / receptor complex / protein C-terminus binding / cell surface receptor signaling pathway / endosome membrane / blood microparticle / protein tyrosine kinase activity / MAPK cascade / immune response / protein dimerization activity / intracellular signal transduction
Immunoglobulin C1-set domain / Furin-like cysteine-rich domain / Immunoglobulin subtype / Immunoglobulin C1-set / Immunoglobulin/major histocompatibility complex, conserved site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase domain / Receptor L-domain / Protein tyrosine kinase / Growth factor receptor domain IV ...Immunoglobulin C1-set domain / Furin-like cysteine-rich domain / Immunoglobulin subtype / Immunoglobulin C1-set / Immunoglobulin/major histocompatibility complex, conserved site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase domain / Receptor L-domain / Protein tyrosine kinase / Growth factor receptor domain IV / Protein kinases ATP-binding region signature. / Immunoglobulin V-set domain / Tyrosine protein kinases specific active-site signature. / Immunoglobulins and major histocompatibility complex proteins signature. / Protein kinase domain profile. / Ig-like domain profile. / Furin-like repeat / Growth factor receptor domain 4 / Tyrosine-protein kinase, catalytic domain / Immunoglobulin-like domain superfamily / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain / Immunoglobulin-like domain / Protein kinase, ATP binding site / Tyrosine protein kinase, EGF/ERB/XmrK receptor / Immunoglobulin-like fold / Immunoglobulin V-set domain / Protein kinase-like domain superfamily / Growth factor receptor cysteine-rich domain superfamily / Tyrosine-protein kinase, active site
Immunoglobulin kappa constant / Receptor tyrosine-protein kinase erbB-2 / Immunoglobulin gamma-1 heavy chain / IGH@ protein
Specimen sourceHomo 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
DateDeposition: Apr 2, 2019 / Release: May 15, 2019Array

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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/peptide , 1 types, 1 molecules A

#1: Protein/peptide Receptor tyrosine-protein kinase 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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Pertuzumab FAB ... , 2 types, 2 molecules BC

#2: Protein/peptide 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: Protein/peptide 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

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Trastuzumab FAB ... , 2 types, 2 molecules DE

#4: Protein/peptide 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: Protein/peptide 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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Non-polymers , 3 types, 9 molecules

#6: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


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


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

Type: COMPLEX

IDNameEntity IDParent-IDSource
1Her2 extracellular domain-Trastuzumab Fab-Pertuzumab Fab complex1, 2, 3, 4, 50MULTIPLE SOURCES
2Human HER2 extracellular domain11RECOMBINANT
3Pertuzumab Fab2, 31RECOMBINANT
4Trastuzumab Fab4, 51RECOMBINANT
Source (natural)

Ncbi tax-ID: 9606 / Organism: Homo sapiens (human)

IDEntity assembly-ID
11
22
33
44
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

Buffer-ID: 1

IDConc.NameFormula
120 mMHEPESC8H18N2O4S
2150 mMSodium ChlorideNaCl
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-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00811809
f_angle_d1.17416110
f_dihedral_angle_d7.0817014
f_chiral_restr0.0571810
f_plane_restr0.0092080

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