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- PDB-6q2n: Cryo-EM structure of RET/GFRa1/GDNF extracellular complex -

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Basic information

Entry
Database: PDB / ID: 6q2n
TitleCryo-EM structure of RET/GFRa1/GDNF extracellular complex
Components
  • GDNF family receptor alpha-1
  • Glial cell line-derived neurotrophic factor
  • Proto-oncogene tyrosine-protein kinase receptor Ret
KeywordsSIGNALING PROTEIN / RET / receptor tyrosine kinase / cryo-EM
Function / homology
Function and homology information


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 / ureteric bud formation / positive regulation of ureteric bud formation / regulation of semaphorin-plexin signaling pathway / postganglionic parasympathetic fiber development / glial cell-derived neurotrophic factor receptor activity / glial cell-derived neurotrophic factor receptor binding ...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 / ureteric bud formation / positive regulation of ureteric bud formation / regulation of semaphorin-plexin signaling pathway / postganglionic parasympathetic fiber development / glial cell-derived neurotrophic factor receptor activity / glial cell-derived neurotrophic factor receptor binding / postsynaptic membrane organization / Peyer's patch morphogenesis / GDF15-GFRAL signaling pathway / positive regulation of metanephric glomerulus development / ureter maturation / embryonic epithelial tube formation / glial cell-derived neurotrophic factor receptor signaling pathway / lymphocyte migration into lymphoid organs / posterior midgut development / regulation of morphogenesis of a branching structure / neurotrophin receptor activity / regulation of dopamine uptake involved in synaptic transmission / Formation of the ureteric bud / membrane protein proteolysis / Formation of the nephric duct / peristalsis / enteric nervous system development / neuron cell-cell adhesion / positive regulation of dopamine secretion / sympathetic nervous system development / positive regulation of branching involved in ureteric bud morphogenesis / organ induction / peripheral nervous system development / plasma membrane protein complex / commissural neuron axon guidance / metanephros development / NCAM1 interactions / neuron maturation / regulation of stem cell differentiation / positive regulation of extrinsic apoptotic signaling pathway in absence of ligand / mRNA stabilization / positive regulation of cell adhesion mediated by integrin / neural crest cell migration / extrinsic component of membrane / ureteric bud development / regulation of axonogenesis / branching involved in ureteric bud morphogenesis / response to pain / homophilic cell-cell adhesion / RET signaling / positive regulation of cell size / embryonic organ development / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / regulation of cell adhesion / cellular response to retinoic acid / NPAS4 regulates expression of target genes / multivesicular body / transmembrane receptor protein tyrosine kinase activity / axon guidance / cell surface receptor protein tyrosine kinase signaling pathway / adult locomotory behavior / growth factor activity / positive regulation of cell differentiation / kidney development / positive regulation of neuron projection development / receptor protein-tyrosine kinase / receptor tyrosine kinase binding / male gonad development / integrin binding / neuron projection development / cell migration / nervous system development / signaling receptor activity / MAPK cascade / regulation of gene expression / RAF/MAP kinase cascade / protein tyrosine kinase activity / negative regulation of neuron apoptotic process / cell surface receptor signaling pathway / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / receptor complex / positive regulation of MAPK cascade / endosome membrane / positive regulation of cell migration / signaling receptor binding / axon / external side of plasma membrane / neuronal cell body / positive regulation of cell population proliferation / calcium ion binding / positive regulation of gene expression / negative regulation of apoptotic process / positive regulation of DNA-templated transcription / Golgi apparatus / signal transduction / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular exosome / extracellular region
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 family / Glial cell line-derived neurotrophic factor receptor / GDNF receptor alpha / 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 family / Glial cell line-derived neurotrophic factor receptor / GDNF receptor alpha / 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 / RET, Cysteine Rich Domain / Transforming growth factor-beta (TGF-beta) family / Transforming growth factor-beta, C-terminal / Transforming growth factor beta like domain / TGF-beta family profile. / Cadherin domain / Cadherin-like / Cadherins domain profile. / 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 / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / 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 / Glial cell line-derived neurotrophic factor / GDNF family receptor alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsLi, J. / Shang, G.J. / Chen, Y.J. / Brautigam, C.A. / Liou, J. / Zhang, X.W. / Bai, X.C.
CitationJournal: Elife / Year: 2019
Title: Cryo-EM analyses reveal the common mechanism and diversification in the activation of RET by different ligands.
Authors: Jie Li / Guijun Shang / Yu-Ju Chen / Chad A Brautigam / Jen Liou / Xuewu Zhang / Xiao-Chen Bai /
Abstract: RET is a receptor tyrosine kinase (RTK) that plays essential roles in development and has been implicated in several human diseases. Different from most of RTKs, RET requires not only its cognate ...RET is a receptor tyrosine kinase (RTK) that plays essential roles in development and has been implicated in several human diseases. Different from most of RTKs, RET requires not only its cognate ligands but also co-receptors for activation, the mechanisms of which remain unclear due to lack of high-resolution structures of the ligand/co-receptor/receptor complexes. Here, we report cryo-EM structures of the extracellular region ternary complexes of GDF15/GFRAL/RET, GDNF/GFRα1/RET, NRTN/GFRα2/RET and ARTN/GFRα3/RET. These structures reveal that all the four ligand/co-receptor pairs, while using different atomic interactions, induce a specific dimerization mode of RET that is poised to bring the two kinase domains into close proximity for cross-phosphorylation. The NRTN/GFRα2/RET dimeric complex further pack into a tetrameric assembly, which is shown by our cell-based assays to regulate the endocytosis of RET. Our analyses therefore reveal both the common mechanism and diversification in the activation of RET by different ligands.
History
DepositionAug 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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Assembly

Deposited unit
A: Glial cell line-derived neurotrophic factor
C: GDNF family receptor alpha-1
E: Proto-oncogene tyrosine-protein kinase receptor Ret
B: Glial cell line-derived neurotrophic factor
D: GDNF family receptor alpha-1
F: Proto-oncogene tyrosine-protein kinase receptor Ret
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,43214
Polymers261,1116
Non-polymers3218
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Glial cell line-derived neurotrophic factor / hGDNF / Astrocyte-derived trophic factor / ATF


Mass: 15096.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GDNF / Production host: Escherichia coli (E. coli) / References: UniProt: P39905
#2: Protein GDNF family receptor alpha-1 / GFR-alpha-1 / RET ligand 1 / TGF-beta-related neurotrophic factor receptor 1


Mass: 46358.551 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GFRA1, GDNFRA, RETL1, TRNR1 / Production host: Homo sapiens (human) / References: UniProt: P56159
#3: Protein Proto-oncogene tyrosine-protein kinase receptor Ret / Cadherin family member 12 / Proto-oncogene c-Ret


Mass: 69100.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RET, CDHF12, CDHR16, PTC, RET51 / Production host: Homo sapiens (human)
References: UniProt: P07949, receptor protein-tyrosine kinase
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: RET, GFRAL and GDF15 extracellular complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 200 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Calibrated magnification: 46729 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37098 / Symmetry type: POINT
Atomic model buildingB value: 190 / Protocol: FLEXIBLE FIT / Space: REAL

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