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- EMDB-2713: Structure of the zebrafish RET tyrosine kinase extracellular domain -

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Basic information

Entry
Database: EMDB / ID: EMD-2713
TitleStructure of the zebrafish RET tyrosine kinase extracellular domain
Map datazTC
Sample
  • Sample: Reconstituted zebrafish RETecd-GDNF-GFRa1 ternary complex
  • Protein or peptide: RET receptor tyrosine kinase
  • Protein or peptide: glial-cell-line-derived neurotrophic factor
  • Protein or peptide: GDNF receptor alpha
Keywordssignal transduction
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
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.
History
DepositionJul 17, 2014-
Header (metadata) releaseJul 23, 2014-
Map releaseOct 1, 2014-
UpdateOct 8, 2014-
Current statusOct 8, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.178
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.178
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2713.png

Downloads & links

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Map

FileDownload / File: emd_2713.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationzTC
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.178 / Movie #1: 0.178
Minimum - Maximum-0.63441044 - 0.75152898
Average (Standard dev.)-0.0003849 (±0.0347409)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 416.64 Å
α=β=γ: 90.0 °

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-0.6340.752-0.000

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Supplemental data

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Sample components

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Entire : Reconstituted zebrafish RETecd-GDNF-GFRa1 ternary complex

EntireName: Reconstituted zebrafish RETecd-GDNF-GFRa1 ternary complex
Components
  • Sample: Reconstituted zebrafish RETecd-GDNF-GFRa1 ternary complex
  • Protein or peptide: RET receptor tyrosine kinase
  • Protein or peptide: glial-cell-line-derived neurotrophic factor
  • Protein or peptide: GDNF receptor alpha

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Supramolecule #1000: Reconstituted zebrafish RETecd-GDNF-GFRa1 ternary complex

SupramoleculeName: Reconstituted zebrafish RETecd-GDNF-GFRa1 ternary complex
type: sample / ID: 1000 / Oligomeric state: Hexamer / Number unique components: 3
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: RET receptor tyrosine kinase

MacromoleculeName: RET receptor tyrosine kinase / type: protein_or_peptide / ID: 1 / Name.synonym: RET / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Danio rerio (zebrafish) / synonym: zebrafish
Molecular weightExperimental: 83 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant cell: sf9

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Macromolecule #2: glial-cell-line-derived neurotrophic factor

MacromoleculeName: glial-cell-line-derived neurotrophic factor / type: protein_or_peptide / ID: 2 / Name.synonym: GDNF / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Danio rerio (zebrafish) / synonym: zebrafish
Recombinant expressionOrganism: unidentified baculovirus / Recombinant cell: sf9

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Macromolecule #3: GDNF receptor alpha

MacromoleculeName: GDNF receptor alpha / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Danio rerio (zebrafish) / synonym: zebrafish
Recombinant expressionOrganism: unidentified baculovirus / Recombinant cell: sf9

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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

Concentration0.03 mg/mL
BufferpH: 8 / Details: 20 mM Tris, 300 mM NaCl, 1 mM Ca++
StainingType: NEGATIVE / Details: 2 % uranyle acetate
GridDetails: quantifoil R1.2/R1.3 coated with a thin carbon layer, glow discharge in air
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
DateMay 18, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 800 / Average electron dose: 100 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 0.9 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles selected manually. 3D starting model consisted of the mTC reconstruction
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: IMAGIC, SPIDER, in-house-software / Number images used: 7510
Final two d classificationNumber classes: 500

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