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- EMDB-6426: Negative-stain electron microscopy of a full-length transmembrane... -

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Basic information

Entry
Database: EMDB / ID: EMD-6426
TitleNegative-stain electron microscopy of a full-length transmembrane receptor tyrosine kinase (PDGFR) in complex with PDGFB
Map dataReconstruction of PDGF-B in complex with full-length PDGFR-Beta
Sample
  • Sample: human PDGF-B bound to PDGFR-Beta
  • Protein or peptide: Platelet-derived growth factor receptor beta
  • Protein or peptide: Platelet-derived growth factor-BB
Keywordsreceptor tyrosine kinase / membrane protein / cancer / signal transduction / phosphorylation
Function / homology
Function and homology information


metanephric glomerular mesangial cell development / positive regulation of vascular associated smooth muscle cell dedifferentiation / platelet-derived growth factor complex / positive regulation of metanephric mesenchymal cell migration / negative regulation of phosphatidylinositol biosynthetic process / platelet activating factor receptor activity / platelet-derived growth factor receptor activity / platelet-derived growth factor beta-receptor activity / cell migration involved in coronary angiogenesis / metanephric glomerular mesangial cell proliferation involved in metanephros development ...metanephric glomerular mesangial cell development / positive regulation of vascular associated smooth muscle cell dedifferentiation / platelet-derived growth factor complex / positive regulation of metanephric mesenchymal cell migration / negative regulation of phosphatidylinositol biosynthetic process / platelet activating factor receptor activity / platelet-derived growth factor receptor activity / platelet-derived growth factor beta-receptor activity / cell migration involved in coronary angiogenesis / metanephric glomerular mesangial cell proliferation involved in metanephros development / positive regulation of glomerular filtration / positive regulation of metanephric mesenchymal cell migration by platelet-derived growth factor receptor-beta signaling pathway / smooth muscle cell chemotaxis / metanephric glomerular capillary formation / cellular response to mycophenolic acid / superoxide-generating NADPH oxidase activator activity / negative regulation of vascular associated smooth muscle cell differentiation / cell migration involved in vasculogenesis / protein kinase C signaling / aorta morphogenesis / positive regulation of hyaluronan biosynthetic process / positive regulation of cell proliferation by VEGF-activated platelet derived growth factor receptor signaling pathway / platelet-derived growth factor binding / retina vasculature development in camera-type eye / vascular endothelial growth factor binding / cardiac myofibril assembly / phosphatidylinositol metabolic process / positive regulation of glomerular mesangial cell proliferation / positive regulation of chemotaxis / interleukin-18-mediated signaling pathway / Signaling by PDGF / paracrine signaling / platelet-derived growth factor receptor binding / positive regulation of vascular associated smooth muscle cell migration / positive regulation of cell-substrate adhesion / positive regulation of DNA biosynthetic process / cellular response to platelet-derived growth factor stimulus / positive regulation of smooth muscle cell migration / positive regulation of calcium ion import / chemoattractant activity / platelet-derived growth factor receptor-beta signaling pathway / monocyte chemotaxis / positive regulation of phosphoprotein phosphatase activity / positive regulation of cell division / negative regulation of platelet activation / platelet-derived growth factor receptor signaling pathway / Non-integrin membrane-ECM interactions / embryonic placenta development / positive regulation of blood vessel endothelial cell migration / positive regulation of phospholipase C activity / positive regulation of calcium-mediated signaling / positive regulation of protein autophosphorylation / positive regulation of vascular associated smooth muscle cell proliferation / collagen binding / reactive oxygen species metabolic process / positive regulation of endothelial cell proliferation / lysosomal lumen / cell chemotaxis / Downstream signal transduction / positive regulation of mitotic nuclear division / negative regulation of miRNA transcription / platelet alpha granule lumen / negative regulation of protein binding / regulation of actin cytoskeleton organization / positive regulation of smooth muscle cell proliferation / growth factor activity / positive regulation of MAP kinase activity / receptor protein-tyrosine kinase / cellular response to growth factor stimulus / response to wounding / Golgi lumen / positive regulation of miRNA transcription / peptidyl-tyrosine phosphorylation / cell surface receptor protein tyrosine kinase signaling pathway / Constitutive Signaling by Aberrant PI3K in Cancer / positive regulation of reactive oxygen species metabolic process / positive regulation of fibroblast proliferation / positive regulation of peptidyl-tyrosine phosphorylation / Platelet degranulation / PIP3 activates AKT signaling / gene expression / heart development / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / cytoplasmic vesicle / RAF/MAP kinase cascade / basolateral plasma membrane / collagen-containing extracellular matrix / protein tyrosine kinase activity / positive regulation of MAPK cascade / protein autophosphorylation / positive regulation of ERK1 and ERK2 cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / receptor complex / protein kinase activity / positive regulation of cell migration / apical plasma membrane / protein heterodimerization activity / endoplasmic reticulum lumen / Golgi membrane / negative regulation of gene expression
Similarity search - Function
Platelet-derived growth factor, N-terminal / Platelet-derived growth factor, N terminal region / Platelet-derived growth factor receptor beta / PDGF/VEGF domain / Platelet-derived growth factor, conserved site / PDGF/VEGF domain / Platelet-derived growth factor (PDGF) family signature. / Platelet-derived growth factor (PDGF) family profile. / Platelet-derived and vascular endothelial growth factors (PDGF, VEGF) family / Tyrosine-protein kinase, receptor class III, conserved site ...Platelet-derived growth factor, N-terminal / Platelet-derived growth factor, N terminal region / Platelet-derived growth factor receptor beta / PDGF/VEGF domain / Platelet-derived growth factor, conserved site / PDGF/VEGF domain / Platelet-derived growth factor (PDGF) family signature. / Platelet-derived growth factor (PDGF) family profile. / Platelet-derived and vascular endothelial growth factors (PDGF, VEGF) family / Tyrosine-protein kinase, receptor class III, conserved site / Receptor tyrosine kinase class III signature. / Cystine-knot cytokine / Immunoglobulin / Immunoglobulin domain / Immunoglobulin domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Immunoglobulin subtype / Immunoglobulin / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Platelet-derived growth factor subunit B / Platelet-derived growth factor receptor beta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 27.0 Å
AuthorsChen PH / Unger VM / He X
CitationJournal: J Mol Biol / Year: 2015
Title: Structure of Full-Length Human PDGFRβ Bound to Its Activating Ligand PDGF-B as Determined by Negative-Stain Electron Microscopy.
Authors: Po-Han Chen / Vinzenz Unger / Xiaolin He /
Abstract: Members of the receptor tyrosine kinases (RTKs) regulate important cellular functions such as cell growth and migration, which are key steps in angiogenesis, in organ morphogenesis and in the ...Members of the receptor tyrosine kinases (RTKs) regulate important cellular functions such as cell growth and migration, which are key steps in angiogenesis, in organ morphogenesis and in the unregulated states, cancer formation. One long-standing puzzle regarding RTKs centers on how the extracellular domain (ECD), which detects and binds to growth factors, is coupled with the intracellular domain kinase activation. While extensive structural works on the soluble portions of RTKs have provided critical insights into RTK structures and functions, lack of a full-length receptor structure has hindered a comprehensive overview of RTK activation. In this study, we successfully purified and determined a 27-Å-resolution structure of PDGFRβ [a full-length human platelet-derived growth factor receptor], in complex with its ligand PDGF-B. In the ligand-stimulated complex, two PDGFRβs assemble into a dimer via an extensive interface essentially running along the full-length of the receptor, suggesting that the membrane-proximal region, the transmembrane helix and the kinase domain of PDGFRβ are involved in dimerization. Major structural differences are seen between the full-length and soluble ECD structures, rationalizing previous experimental data on how membrane-proximal domains modulate receptor ligand-binding affinity and dimerization efficiency. Also, in contrast to the 2-fold symmetry of the ECD, the intracellular kinase domains adopt an asymmetric dimer arrangement, in agreement with prior observations for the closely related KIT receptor. In essence, the structure provides a first glimpse into how platelet-derived growth factor receptor ECD, upon ligand stimulation, is coupled to its intracellular domain kinase activation.
History
DepositionAug 12, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseMay 4, 2016-
UpdateMay 4, 2016-
Current statusMay 4, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6426.png

Downloads & links

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Map

FileDownload / File: emd_6426.map.gz / Format: CCP4 / Size: 5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of PDGF-B in complex with full-length PDGFR-Beta
Voxel sizeX=Y=Z: 3.71 Å
Density
Contour LevelBy EMDB: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.1818473 - 0.30766541
Average (Standard dev.)0.00180476 (±0.01717539)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions110110110
Spacing110110110
CellA=B=C: 408.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.713.713.71
M x/y/z110110110
origin x/y/z0.0000.0000.000
length x/y/z408.100408.100408.100
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-31-64
NX/NY/NZ6963129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS110110110
D min/max/mean-0.1820.3080.002

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Supplemental data

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Sample components

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Entire : human PDGF-B bound to PDGFR-Beta

EntireName: human PDGF-B bound to PDGFR-Beta
Components
  • Sample: human PDGF-B bound to PDGFR-Beta
  • Protein or peptide: Platelet-derived growth factor receptor beta
  • Protein or peptide: Platelet-derived growth factor-BB

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Supramolecule #1000: human PDGF-B bound to PDGFR-Beta

SupramoleculeName: human PDGF-B bound to PDGFR-Beta / type: sample / ID: 1000
Details: The sample is cross-linked via the GraFix method after the gel filtration step.
Oligomeric state: a PDGF-B dimer bound to two PDGFR-Beta / Number unique components: 2
Molecular weightExperimental: 300 KDa / Theoretical: 300 KDa / Method: gel filtration and SDS-PAGE analysis

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Macromolecule #1: Platelet-derived growth factor receptor beta

MacromoleculeName: Platelet-derived growth factor receptor beta / type: protein_or_peptide / ID: 1 / Name.synonym: PDGFR-Beta
Details: N-terminal Flag tag is attached after a secretion signal derived from Gaussia luciferase.
Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: plasma membrane
Molecular weightExperimental: 150 KDa / Theoretical: 150 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293GnTI- / Recombinant plasmid: BacMam
SequenceUniProtKB: Platelet-derived growth factor receptor beta

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Macromolecule #2: Platelet-derived growth factor-BB

MacromoleculeName: Platelet-derived growth factor-BB / type: protein_or_peptide / ID: 2 / Name.synonym: PDGF-BB / Details: C-terminal residues 191-241 removed / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Extracellular
Molecular weightExperimental: 12 KDa / Theoretical: 12 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293GnTI- / Recombinant plasmid: BacMam
SequenceUniProtKB: Platelet-derived growth factor subunit B

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.6
Details: 150 mM NaCl, 10 mM HEPES, 0.01% LMNG, ~20% glycerol
StainingType: NEGATIVE
Details: Grids with adsorbed protein were floated on 0.75% w/v uranyl formate for 20 seconds.
GridDetails: 400 mesh copper grid with ~2.1 nm thin carbon support, glow-discharged in ambient air atmosphere
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeOTHER
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.4 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 50
DateDec 1, 2014
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Number real images: 100
Tilt angle min0

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: OTHER / Software - Name: XMIPP, RELION / Number images used: 4234
DetailsInitial models are reconstructed by RCT. Visual inspection of similar classes were pooled, and one initial model was used as an input model for 3D classification into three classes using RELION.

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