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- EMDB-2712: Structure of the RET receptor tyrosine kinase extracellular domain -

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Entry
Database: EMDB / ID: 2712
TitleStructure of the RET receptor tyrosine kinase extracellular domain
Map dataReconstruction of a reconstituted mammalian RETecd-GDNF-GFRa1 ternary (mTC) complex
SampleReconstituted mammalian RETecd-GDNF-GFRa1 ternary complex
  • RET receptor tyrosine kinase
  • GDNF receptor alphaGFRα
  • glial-cell-line-derived neurotrophic factor
Keywordsvertebrate development / human diseases / RET-GFL-GFRa complex
Function / homologyCadherin domain / Glial cell line-derived neurotrophic factor receptor, alpha 1 / Protein kinases ATP-binding region signature. / Protein tyrosine kinase / Tyrosine protein kinases specific active-site signature. / Protein kinase domain / Serine-threonine/tyrosine-protein kinase, catalytic domain / Transforming growth factor-beta, C-terminal / Cadherin-like / Protein kinase domain profile. ...Cadherin domain / Glial cell line-derived neurotrophic factor receptor, alpha 1 / Protein kinases ATP-binding region signature. / Protein tyrosine kinase / Tyrosine protein kinases specific active-site signature. / Protein kinase domain / Serine-threonine/tyrosine-protein kinase, catalytic domain / Transforming growth factor-beta, C-terminal / Cadherin-like / Protein kinase domain profile. / GDNF/GAS1 domain / TGF-beta family profile. / Glial cell line-derived neurotrophic factor receptor / NCAM1 interactions / RAF/MAP kinase cascade / RET signaling / Cadherins domain profile. / Tyrosine-protein kinase, active site / Protein kinase, ATP binding site / Protein kinase-like domain superfamily / Transforming growth factor beta like domain / GDNF receptor alpha / Cystine-knot cytokine / Tyrosine-protein kinase, catalytic domain / Glial cell line-derived neurotrophic factor receptor, alpha 1/2 / Glial cell line-derived neurotrophic factor / Tyrosine-protein kinase, Ret receptor / GDNF/GAS1 / Cadherin-like superfamily / Glial cell line-derived neurotrophic factor-like / postsynaptic membrane organization / chemoattractant activity involved in axon guidance / mesenchymal to epithelial transition involved in metanephros morphogenesis / dorsal spinal cord development / positive regulation of mesenchymal to epithelial transition involved in metanephros morphogenesis / postganglionic parasympathetic fiber development / positive regulation of ureteric bud formation / regulation of dopamine uptake involved in synaptic transmission / ureteric bud formation / regulation of semaphorin-plexin signaling pathway / embryonic epithelial tube formation / lymphocyte migration into lymphoid organs / glial cell-derived neurotrophic factor receptor signaling pathway / regulation of stem cell differentiation / positive regulation of metanephric glomerulus development / Peyer's patch morphogenesis / posterior midgut development / enteric nervous system development / ureter maturation / membrane protein proteolysis / peristalsis / positive regulation of monooxygenase activity / positive regulation of dopamine secretion / regulation of morphogenesis of a branching structure / positive regulation of peptidyl-serine phosphorylation of STAT protein / neurotrophin receptor activity / neuron cell-cell adhesion / innervation / plasma membrane protein complex / sympathetic nervous system development / positive regulation of neuron maturation / positive regulation of branching involved in ureteric bud morphogenesis / neuron maturation / metanephros development / positive regulation of extrinsic apoptotic signaling pathway in absence of ligand / mRNA stabilization / commissural neuron axon guidance / positive regulation of cell adhesion mediated by integrin / neurogenesis / positive regulation of cell size / neural crest cell migration / organ induction / response to pain / embryonic organ development / ureteric bud development / regulation of axonogenesis / branching involved in ureteric bud morphogenesis / homophilic cell adhesion via plasma membrane adhesion molecules / regulation of cell adhesion / retina development in camera-type eye / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / adult locomotory behavior / anchored component of membrane / cellular response to retinoic acid / positive regulation of neuron projection development / positive regulation of cell differentiation / neuron differentiation / receptor protein-tyrosine kinase / transmembrane receptor protein tyrosine kinase activity / neuron projection development / activation of cysteine-type endopeptidase activity involved in apoptotic process / nervous system development / growth factor activity / male gonad development / axon guidance / Ras guanyl-nucleotide exchange factor activity / kidney development / positive regulation of peptidyl-tyrosine phosphorylation / integrin binding / transmembrane receptor protein tyrosine kinase signaling pathway
Function and homology information
SourceHomo sapiens (human) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / 24 Å resolution
AuthorsGoodman K / Kjaer S / Beuron F / Knowles P / Nawrotek A / Burns E / Purkiss A / George R / Santoro M / Morris EP / McDonald NQ
CitationJournal: Cell Rep / Year: 2014
Title: RET recognition of GDNF-GFRα1 ligand by a composite binding site promotes membrane-proximal self-association.
Authors: Kerry M Goodman / Svend Kjær / Fabienne Beuron / Phillip P Knowles / Agata Nawrotek / Emily M Burns / Andrew G Purkiss / Roger George / Massimo Santoro / Edward P Morris / Neil Q McDonald
Abstract: The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a ...The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.
Validation ReportPDB-ID: 4ux8

SummaryFull reportAbout validation report
DateDeposition: Jul 17, 2014 / Header (metadata) release: Jul 23, 2014 / Map release: Oct 1, 2014 / Last update: Oct 8, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.42
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.42
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-4ux8
  • Surface level: 2.42
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2712.map.gz (map file in CCP4 format, 3457 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
96 pix
4.34 Å/pix.
= 416.64 Å
96 pix
4.34 Å/pix.
= 416.64 Å
96 pix
4.34 Å/pix.
= 416.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.34 Å
Density
Contour Level:2.42 (by author), 2.42 (movie #1):
Minimum - Maximum-19.55950928 - 26.86942863
Average (Standard dev.)0E-8 (1.00000000)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions969696
Origin-48-48-48
Limit474747
Spacing969696
CellA=B=C: 416.64 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.344.344.34
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z416.640416.640416.640
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-19.56026.869-0.000

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Supplemental data

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Sample components

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Entire Reconstituted mammalian RETecd-GDNF-GFRa1 ternary complex

EntireName: Reconstituted mammalian RETecd-GDNF-GFRa1 ternary complex
Details: Monodisperse. Measured mass difference with theoretical MW corresponds to glycosylation.
Number of components: 3 / Oligomeric State: Hexamer
MassTheoretical: 260 kDa / Experimental: 340 kDa / Measured by: SEC-MALS

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Component #1: protein, RET receptor tyrosine kinase

ProteinName: RET receptor tyrosine kinase / a.k.a: RET / Number of Copies: 2 / Recombinant expression: Yes
MassTheoretical: 80 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Chinese Hamster Ovary / Strain: Lec8

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Component #2: protein, GDNF receptor alpha

ProteinName: GDNF receptor alphaGFRα / Recombinant expression: Yes / Number of Copies: 2
MassTheoretical: 40 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Chinese Hamster Ovary

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Component #3: protein, glial-cell-line-derived neurotrophic factor

ProteinName: glial-cell-line-derived neurotrophic factor / a.k.a: GDNF / Details: from Amgen (USA) / Number of Copies: 2 / Recombinant expression: Yes
MassTheoretical: 10 kDa
SourceSpecies: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.03 mg/ml / Buffer solution: 20 mM Tris HCl, 300 mM NaCl, 1 mM Ca++ / pH: 8
Support filmquantifoil (R1.2/1.3) coated with a thin carbon layer, glow discharge in air.
StainingSamples were applied to glow-discharged grids and stained with 2% uranyl acetate
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Nov 8, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 100 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 900 - nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: TVIPS TEMCAM-F415 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1200 / Bit depth: 16

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 975 / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 8519
Details: The particles were selected manually. The starting model was calculated from reference-free classes using angular reconstitution methods and further refined by projection matching.
3D reconstructionAlgorithm: angular reconstitution / Software: IMAGIC, SPIDER, in-house, software / Resolution: 24 Å / Resolution method: FSC 0.5, semi-independent

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