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

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Basic information

Entry
Database: PDB / ID: 7aml
TitleRET/GDNF/GFRa1 extracellular complex Cryo-EM structure
Components
  • GDNF family receptor alphaGFRα
  • Glial cell line-derived neurotrophic factor
  • Proto-oncogene tyrosine-protein kinase receptor Ret
KeywordsSIGNALING PROTEIN / VERTEBRATE DEVELOPMENT / PART OF THE RET-GFL-GFRA COMPLEX / NEUROTROPHIC FACTOR
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 protein-tyrosine kinase / receptor tyrosine kinase binding / cell surface receptor protein tyrosine kinase signaling pathway / neuron projection development / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsAdams, S.E. / Earl, C.P. / Purkiss, A.G. / McDonald, N.Q.
Funding support United Kingdom, 1items
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, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 14, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
A: Proto-oncogene tyrosine-protein kinase receptor Ret
D: Proto-oncogene tyrosine-protein kinase receptor Ret
B: GDNF family receptor alpha
C: Glial cell line-derived neurotrophic factor
E: GDNF family receptor alpha
F: Glial cell line-derived neurotrophic factor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)245,53830
Polymers241,6786
Non-polymers3,86024
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, homology
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "D"
d_2ens_1chain "A"
d_1ens_2chain "B"
d_2ens_2chain "E"
d_1ens_3chain "C"
d_2ens_3chain "F"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLYPROL1 - 596
d_12ens_1NAGNAGM
d_13ens_1NAGNAGN
d_14ens_1NAGNAGO
d_15ens_1NAGNAGP
d_16ens_1NAGNAGQ
d_17ens_1NAGNAGR
d_18ens_1NAGNAGS
d_21ens_1GLYPROA1 - 596
d_22ens_1NAGNAGB
d_23ens_1NAGNAGC
d_24ens_1NAGNAGD
d_25ens_1NAGNAGE
d_26ens_1NAGNAGF
d_27ens_1NAGNAGG
d_28ens_1NAGNAGH
d_11ens_2ARGGLYI1 - 304
d_21ens_2ARGGLYT1 - 304
d_11ens_3GLNVALJ1 - 98
d_12ens_3NAGNAGK
d_21ens_3GLNVALU1 - 98
d_22ens_3NAGNAGV

NCS ensembles :
ID
ens_1
ens_2
ens_3

NCS oper:
IDCodeMatrixVector
1given(-0.999998415374, -0.00177998617107), (0.00177998726659, -0.999998415153), (0.999999998882)179.366558691, 165.967286175, -0.179739366316
2given(-0.999720161988, -0.00942115824473, -0.0216988362036), (0.00923448692586, -0.999919626719, 0.00868702211368), (-0.021778934007, 0.00848421353545, 0.999726810761)181.27484087, 165.214413934, 0.41746535266
3given(-0.999989199402, 0.00464761817397), (0.000133492566125, -0.999736963592, 0.0229343804838), (0.00464577856382, 0.0229347532013, 0.999726169427)179.221881202, 165.271640213, -2.39670297898

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Components

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


Mass: 69536.805 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: ret, ret1 / Plasmid: pBacPAK-LL-zRETECD-3C-Protein-A / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-Sf21-AE
References: UniProt: A8E7C6, receptor protein-tyrosine kinase
#2: Protein GDNF family receptor alpha / GFRα


Mass: 39735.168 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: gfra1a, gfralpha1a / Plasmid: pBacPAK-LL-zGFRa1aD1-D3-3C-ProteinA / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-Sf21-AE / References: UniProt: Q98TT9
#3: Protein Glial cell line-derived neurotrophic factor / / zGDNF


Mass: 11567.194 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: gdnf / Plasmid: pBacPAK-LL-melittin-zGDNFmat-3C-ProteinA / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-Sf21-AE / References: UniProt: Q98TU0
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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

ComponentName: Ternary complex of RET extracellular domain with GDNF and GFRa1 ligand-coreceptor pair
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: NO
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: IPLB-Sf21-AE / Plasmid: pBacPAK
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris-HClTris(HOCH2)3CNH21
2150 mMsodium chlorideNaClSodium chloride1
31 mMcalcium chlorideCaCL21
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Crosslinked using the GraFIX method with a gradient of 0-0.1% glutaraldehyde, 5-20% sucrose. Further purified by size exclusion chromatography.
Specimen supportDetails: 45 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: 90 s wait time, 5 s blot time

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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: 46296 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1400 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 48.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 12475
Details: 6105 movies were collected without stage tilt, 6375 movies were collected with a tilt angle of 30 degrees.
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1RELIONparticle selection
2Gautomatchparticle selection
3EPUimage acquisition
5CTFFIND4.1CTF correction
8PHENIX1.18.2model fitting
10RELIONinitial Euler assignment
11cryoSPARC2final Euler assignment
12cryoSPARC2classification
13cryoSPARC23D reconstruction
20PHENIX1.18.2model refinement
21Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2549540
Details: 1156517 particles picked from 0 tilt data, 1393023 particles picked from 30-degree tilt data
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 382547 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7AB8

7ab8

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 37.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00516330
ELECTRON MICROSCOPYf_angle_d0.81922104
ELECTRON MICROSCOPYf_dihedral_angle_d23.6296156
ELECTRON MICROSCOPYf_chiral_restr0.0572486
ELECTRON MICROSCOPYf_plane_restr0.0042872
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.303118945511
ens_2d_2AELECTRON MICROSCOPYNCS constraints0.0448414218699
ens_3d_2AELECTRON MICROSCOPYNCS constraints0.422740336666

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