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- EMDB-11822: RET/GDNF/GFRa1 extracellular complex Cryo-EM structure -

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Basic information

Entry
Database: EMDB / ID: EMD-11822
TitleRET/GDNF/GFRa1 extracellular complex Cryo-EM structure
Map dataSharpened map
Sample
  • Complex: Ternary complex of RET extracellular domain with GDNF and GFRa1 ligand-coreceptor pair
    • Protein or peptide: Proto-oncogene tyrosine-protein kinase receptor Ret
    • Protein or peptide: GDNF family receptor alphaGFRα
    • Protein or peptide: Glial cell line-derived neurotrophic factor
  • Ligand: CALCIUM IONCalcium
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


branchiomeric skeletal muscle development / pronephros morphogenesis / RAF/MAP kinase cascade / : / diencephalon development / positive regulation of ureteric bud formation / postganglionic parasympathetic fiber development / positive regulation of monooxygenase activity / regulation of dopaminergic neuron differentiation / glial cell-derived neurotrophic factor receptor binding ...branchiomeric skeletal muscle development / pronephros morphogenesis / RAF/MAP kinase cascade / : / diencephalon development / positive regulation of ureteric bud formation / postganglionic parasympathetic fiber development / positive regulation of monooxygenase activity / regulation of dopaminergic neuron differentiation / glial cell-derived neurotrophic factor receptor binding / regulation of morphogenesis of a branching structure / regulation of dopamine uptake involved in synaptic transmission / enteric nervous system development / neural crest cell migration involved in autonomic nervous system development / positive regulation of branching involved in ureteric bud morphogenesis / peristalsis / sympathetic nervous system development / peripheral nervous system development / mRNA stabilization / metanephros development / axon extension / positive regulation of kinase activity / neural crest cell migration / branching involved in ureteric bud morphogenesis / homophilic cell adhesion via plasma membrane adhesion molecules / MAP kinase kinase kinase activity / transmembrane receptor protein tyrosine kinase activity / growth factor activity / receptor tyrosine kinase binding / receptor protein-tyrosine kinase / neuron projection development / cell surface receptor protein tyrosine kinase signaling pathway / signaling receptor activity / nervous system development / protein-containing complex assembly / negative regulation of neuron apoptotic process / receptor complex / membrane raft / axon / external side of plasma membrane / calcium ion binding / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular region / ATP binding / plasma membrane
Similarity search - Function
: / Glial cell line-derived neurotrophic factor receptor, alpha 1 / Glial cell line-derived neurotrophic factor / Glial cell line-derived neurotrophic factor receptor, alpha 1/2 / Glial cell line-derived neurotrophic factor receptor / GDNF receptor alpha / Glial cell line-derived neurotrophic factor family / GDNF/GAS1 / GDNF/GAS1 domain / GDNF/GAS1 domain ...: / Glial cell line-derived neurotrophic factor receptor, alpha 1 / Glial cell line-derived neurotrophic factor / Glial cell line-derived neurotrophic factor receptor, alpha 1/2 / Glial cell line-derived neurotrophic factor receptor / GDNF receptor alpha / Glial cell line-derived neurotrophic factor family / GDNF/GAS1 / GDNF/GAS1 domain / GDNF/GAS1 domain / Tyrosine-protein kinase, Ret receptor / Tyrosine-protein kinase receptor Ret, cadherin like domain 3 / Ret, cadherin like domain 1 / RET, cadherin-like domain 4 / RET Cadherin like domain 1 / RET Cadherin like domain 3 / RET Cadherin like domain 4 / Transforming growth factor-beta, C-terminal / Transforming growth factor beta like domain / TGF-beta family profile. / Cadherin repeats. / Cadherins domain profile. / Cadherin-like / Cadherin-like superfamily / Cystine-knot cytokine / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Proto-oncogene tyrosine-protein kinase receptor Ret / GDNF family receptor alpha / Glial cell line-derived neurotrophic factor
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsAdams SE / Earl CP / Purkiss AG / McDonald NQ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
The Francis Crick Institute10115 United Kingdom
CitationJournal: Structure / Year: 2021
Title: A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing.
Authors: Sarah E Adams / Andrew G Purkiss / Phillip P Knowles / Andrea Nans / David C Briggs / Annabel Borg / Christopher P Earl / Kerry M Goodman / Agata Nawrotek / Aaron J Borg / Pauline B McIntosh ...Authors: Sarah E Adams / Andrew G Purkiss / Phillip P Knowles / Andrea Nans / David C Briggs / Annabel Borg / Christopher P Earl / Kerry M Goodman / Agata Nawrotek / Aaron J Borg / Pauline B McIntosh / Francesca M Houghton / Svend Kjær / Neil Q McDonald /
Abstract: RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET ...RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RET-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RET-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.
History
DepositionOct 9, 2020-
Header (metadata) releaseJan 13, 2021-
Map releaseJan 13, 2021-
UpdateJul 14, 2021-
Current statusJul 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.269
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.269
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7aml
  • Surface level: 0.269
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7aml
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11822.png

Downloads & links

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Map

FileDownload / File: emd_11822.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.269 / Movie #1: 0.269
Minimum - Maximum-2.3209963 - 3.551591
Average (Standard dev.)0.0029890374 (±0.048600234)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ540540540
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-2.3213.5520.003

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Supplemental data

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Mask #1

Fileemd_11822_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_11822_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Intermediate symmetry expansion map, unsharpened

Fileemd_11822_additional_2.map
AnnotationIntermediate symmetry expansion map, unsharpened
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Intermediate symmetry expansion map, sharpened

Fileemd_11822_additional_3.map
AnnotationIntermediate symmetry expansion map, sharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_11822_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_11822_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Ternary complex of RET extracellular domain with GDNF and GFRa1 l...

EntireName: Ternary complex of RET extracellular domain with GDNF and GFRa1 ligand-coreceptor pair
Components
  • Complex: Ternary complex of RET extracellular domain with GDNF and GFRa1 ligand-coreceptor pair
    • Protein or peptide: Proto-oncogene tyrosine-protein kinase receptor Ret
    • Protein or peptide: GDNF family receptor alphaGFRα
    • Protein or peptide: Glial cell line-derived neurotrophic factor
  • Ligand: CALCIUM IONCalcium
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Ternary complex of RET extracellular domain with GDNF and GFRa1 l...

SupramoleculeName: Ternary complex of RET extracellular domain with GDNF and GFRa1 ligand-coreceptor pair
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Danio rerio (zebrafish)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: IPLB-Sf21-AE / Recombinant plasmid: pBacPAK
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: Proto-oncogene tyrosine-protein kinase receptor Ret

MacromoleculeName: Proto-oncogene tyrosine-protein kinase receptor Ret / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 69.536805 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GLYFPQRLYT ENIYVGQQQG SPLLQVISMR EFPTERPYFF LCSHRDAFTS WFHIDEASGV LYLNKTLEWS DFSSLRSGSV RSPKDLTLK VGVSSTPPMK VMCTILPTVE VKLSFINDTA PSCGQVELST LCFPEKISNP HITENREPGA LRQLRRFTHM S ICPNYTIS ...String:
GLYFPQRLYT ENIYVGQQQG SPLLQVISMR EFPTERPYFF LCSHRDAFTS WFHIDEASGV LYLNKTLEWS DFSSLRSGSV RSPKDLTLK VGVSSTPPMK VMCTILPTVE VKLSFINDTA PSCGQVELST LCFPEKISNP HITENREPGA LRQLRRFTHM S ICPNYTIS YGVVAGSSVP FAVDDSTSEL VVTAQVDREE KEVYHLDIVC MVRTERNLEE VFRSLHVNIY DEDDNSPYVN GT DTEDVLV EFDRSEGTVF GTLFVYDRDT TPVYPTNQVQ NKLVGTLMTN DSWIKNNFAI EHKFREEKAI FGNVRGTVHE YKL KLSQNL SVTEQRSFLL GYLVNDTTFP GPEGTVLLHF NVTVLPVPIR FSNVTYSFTV SQKATTYSQI GKVCVENCQK FKGI DVTYQ LEIVDRNITA EAQSCYWAVS LAQNPNDNTG VLYVNDTKVL RRPECQELEY VVIAQEQQNK LQAKTQLTVS FQGEA DSLR TDEPRFPACA EKRQRGDCEA TRGLGAPTGR CQWRQGRDKG ISKRYSTCSP NLGTCPDGYC DAIESKNISI CPQDCS SEA IIGGYERDLY GIKAGHGTCY CFEGKCFCER DEPEDDICND MCGSEFENLY FQ

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Macromolecule #2: GDNF family receptor alpha

MacromoleculeName: GDNF family receptor alpha / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 39.735168 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EESYFSSSNR LDCVKANELC LKEPGCSSKY RTMRQCVAGR ESNFSMATGM EAKDECRLVL DALKQSPLYN CRCKRGMKKE KNCLRIYWG IYQHLQGNDL LEDSPYEPVN SRLSDIFRLA PIYSGEPALA KENNCLNAAK ACNLNDTCKK YRSAYISPCT S RVSTAEVC ...String:
EESYFSSSNR LDCVKANELC LKEPGCSSKY RTMRQCVAGR ESNFSMATGM EAKDECRLVL DALKQSPLYN CRCKRGMKKE KNCLRIYWG IYQHLQGNDL LEDSPYEPVN SRLSDIFRLA PIYSGEPALA KENNCLNAAK ACNLNDTCKK YRSAYISPCT S RVSTAEVC NKRKCHKALR QFFDKVPPKH SYGMLYCSCP LGDQSACSER RRQTIVPACS YEDKERPNCL TLQVSCKTNY IC RSRLADF FTNCQPEPLS LSGCLKENYA DCLLSYSGLI GTVMTPNYLR SPKISVSPFC DCSSSGNSKE ECDRFTEFFT DNA CLRNAI QAFGNGGSEF LEVLFQGPGG GENLYFQ

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Macromolecule #3: Glial cell line-derived neurotrophic factor

MacromoleculeName: Glial cell line-derived neurotrophic factor / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 11.567194 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
VKGQGRGCLL KEIHLNVTDL DLGYRTKEEL IFRYCSGPCH DAETNYDKIL NNLTHNKKLD KDTPSRTCCR PIAFDDDISF LDDSLEYHT LKKHSAKKCA CV

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 8 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 16 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2tris-HClTris
150.0 mMNaClSodium chloridesodium chloride
1.0 mMCaCL2calcium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 45 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK II / Details: 90 s wait time, 5 s blot time.
DetailsCrosslinked using the GraFIX method with a gradient of 0-0.1% glutaraldehyde, 5-20% sucrose. Further purified by size exclusion chromatography.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 46296
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 12475 / Average exposure time: 9.0 sec. / Average electron dose: 48.6 e/Å2
Details: 6105 movies were collected without stage tilt, 6375 movies were collected with a tilt angle of 30 degrees.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2549540
Details: 1156517 particles picked from 0 tilt data, 1393023 particles picked from 30-degree tilt data
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: INSILICO MODEL / In silico model: ab initio model generated in cryoSPARC2
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 200 / Avg.num./class: 1913 / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 382547
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7ab8

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7aml:
RET/GDNF/GFRa1 extracellular complex Cryo-EM structure

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