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- EMDB-20579: Cryo-EM structure of RET/GFRa3/ARTN extracellular complex. The 3D... -

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Entry
Database: EMDB / ID: EMD-20579
TitleCryo-EM structure of RET/GFRa3/ARTN extracellular complex. The 3D refinement was applied with C2 symmetry
Map dataCryo-EM structure of RET/GFRa3/ARTN extracellular complex. The 3D refinement was applied with C2 symmetry.
Sample
  • Complex: RET, GFRAL and GDF15 extracellular complex
    • Protein or peptide: Ubiquitin-like protein SMT3,Artemin
    • Protein or peptide: GDNF family receptor alpha-3
    • Protein or peptide: Proto-oncogene tyrosine-protein kinase receptor Ret
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
KeywordsRET / receptor tyrosine kinase / cryo-EM / SIGNALING PROTEIN
Function / homology
Function and homology information


glial cell-derived neurotrophic factor receptor activity / glial cell-derived neurotrophic factor receptor binding / 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 ...glial cell-derived neurotrophic factor receptor activity / glial cell-derived neurotrophic factor receptor binding / 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 / axon guidance receptor activity / SUMO is conjugated to E1 (UBA2:SAE1) / membrane protein proteolysis / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / Formation of the ureteric bud / SUMO is proteolytically processed / SUMOylation of transcription factors / positive regulation of neuron maturation / SUMOylation of transcription cofactors / Postmitotic nuclear pore complex (NPC) reformation / neuron cell-cell adhesion / Formation of the nephric duct / septin ring / SUMOylation of DNA damage response and repair proteins / enteric nervous system development / SUMOylation of DNA replication proteins / sympathetic nervous system development / induction of positive chemotaxis / peripheral nervous system development / SUMOylation of SUMOylation proteins / innervation / plasma membrane protein complex / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / positive regulation of extrinsic apoptotic signaling pathway in absence of ligand / neuron maturation / NCAM1 interactions / SUMOylation of RNA binding proteins / positive regulation of cell adhesion mediated by integrin / neural crest cell migration / SUMOylation of chromatin organization proteins / extrinsic component of membrane / ureteric bud development / regulation of axonogenesis / response to pain / homophilic cell adhesion via plasma membrane adhesion molecules / ubiquitin-like protein ligase binding / RET signaling / protein sumoylation / neuroblast proliferation / positive regulation of cell size / regulation of cell adhesion / cellular response to retinoic acid / transmembrane receptor protein tyrosine kinase activity / NPAS4 regulates expression of target genes / axon guidance / : / condensed nuclear chromosome / growth factor activity / neuron migration / placental growth factor receptor activity / insulin receptor activity / vascular endothelial growth factor receptor activity / hepatocyte growth factor receptor activity / macrophage colony-stimulating factor receptor activity / platelet-derived growth factor alpha-receptor activity / platelet-derived growth factor beta-receptor activity / stem cell factor receptor activity / boss receptor activity / protein tyrosine kinase collagen receptor activity / brain-derived neurotrophic factor receptor activity / GPI-linked ephrin receptor activity / transmembrane-ephrin receptor activity / epidermal growth factor receptor activity / fibroblast growth factor receptor activity / insulin-like growth factor receptor activity / receptor protein-tyrosine kinase / receptor tyrosine kinase binding / positive regulation of neuron projection development / protein tag activity / cell surface receptor protein tyrosine kinase signaling pathway / MAPK cascade / signaling receptor activity / nervous system development / retina development in camera-type eye / protein tyrosine kinase activity / RAF/MAP kinase cascade / histone H3Y41 kinase activity / histone H2AXY142 kinase activity / positive regulation of MAPK cascade / early endosome / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / receptor complex / endosome membrane / positive regulation of cell migration / protein phosphorylation / response to xenobiotic stimulus / axon / external side of plasma membrane / signaling receptor binding
Similarity search - Function
Glial cell line-derived neurotrophic factor receptor, alpha 3 / 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 ...Glial cell line-derived neurotrophic factor receptor, alpha 3 / 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 / RET, Cysteine Rich Domain / Transforming growth factor-beta (TGF-beta) family / Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / 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 / : / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / 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 / Ubiquitin-like domain superfamily / 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
GDNF family receptor alpha-3 / Proto-oncogene tyrosine-protein kinase receptor Ret / Ubiquitin-like protein SMT3 / Artemin
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLi J / Shang GJ
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, 2019-
Header (metadata) releaseOct 2, 2019-
Map releaseOct 2, 2019-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6q2s
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20579.png

Downloads & links

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Map

FileDownload / File: emd_20579.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of RET/GFRa3/ARTN extracellular complex. The 3D refinement was applied with C2 symmetry.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 240 pix.
= 256.8 Å		1.07 Å/pix.
x 240 pix.
= 256.8 Å		1.07 Å/pix.
x 240 pix.
= 256.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.036 / Movie #1: 0.036
Minimum - Maximum-0.09402748 - 0.17578915
Average (Standard dev.)0.0009606698 (±0.007141335)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 256.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z256.800256.800256.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0940.1760.001

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Supplemental data

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Sample components

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Entire : RET, GFRAL and GDF15 extracellular complex

EntireName: RET, GFRAL and GDF15 extracellular complex
Components
  • Complex: RET, GFRAL and GDF15 extracellular complex
    • Protein or peptide: Ubiquitin-like protein SMT3,Artemin
    • Protein or peptide: GDNF family receptor alpha-3
    • Protein or peptide: Proto-oncogene tyrosine-protein kinase receptor Ret
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION

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Supramolecule #1: RET, GFRAL and GDF15 extracellular complex

SupramoleculeName: RET, GFRAL and GDF15 extracellular complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 200 kDa/nm

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Macromolecule #1: Ubiquitin-like protein SMT3,Artemin

MacromoleculeName: Ubiquitin-like protein SMT3,Artemin / type: protein_or_peptide / ID: 1
Details: A SUMO protein was fused to the N-terminal of NRTN for the protein expression
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.793287 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMSDSEVN QEAKPEVKPE VKPETHINLK VSDGSSEIFF KIKKTTPLRR LMEAFAKRQG KEMDSLRFL YDGIRIQADQ TPEDLDMEDN DIIEAHREQI GGSAGGPGSR ARAAGARGCR LRSQLVPVRA LGLGHRSDEL V RFRFCSGS ...String:
MGSSHHHHHH SSGLVPRGSH MASMSDSEVN QEAKPEVKPE VKPETHINLK VSDGSSEIFF KIKKTTPLRR LMEAFAKRQG KEMDSLRFL YDGIRIQADQ TPEDLDMEDN DIIEAHREQI GGSAGGPGSR ARAAGARGCR LRSQLVPVRA LGLGHRSDEL V RFRFCSGS CRRARSPHDL SLASLLGAGA LRPPPGSRPV SQPCCRPTRY EAVSFMDVNS TWRTVDRLSA TACGCLG

UniProtKB: Ubiquitin-like protein SMT3, Artemin

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Macromolecule #2: GDNF family receptor alpha-3

MacromoleculeName: GDNF family receptor alpha-3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 38.370023 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DPLPTESRLM NSCLQARRKC QADPTCSAAY HHLDSCTSSI STPLPSEEPS VPADCLEAAQ QLRNSSLIGC MCHRRMKNQV ACLDIYWTV HRARSLGNYE LDVSPYEDTV TSKPWKMNLS KLNMLKPDSD LCLKFAMLCT LNDKCDRLRK AYGEACSGPH C QRHVCLRQ ...String:
DPLPTESRLM NSCLQARRKC QADPTCSAAY HHLDSCTSSI STPLPSEEPS VPADCLEAAQ QLRNSSLIGC MCHRRMKNQV ACLDIYWTV HRARSLGNYE LDVSPYEDTV TSKPWKMNLS KLNMLKPDSD LCLKFAMLCT LNDKCDRLRK AYGEACSGPH C QRHVCLRQ LLTFFEKAAE PHAQGLLLCP CAPNDRGCGE RRRNTIAPNC ALPPVAPNCL ELRRLCFSDP LCRSRLVDFQ TH CHPMDIL GTCATEQSRC LRAYLGLIGT AMTPNFVSNV NTSVALSCTC RGSGNLQEEC EMLEGFFSHN PCLTEAIAAK MRF HSQLFS QDWPHPGTHH HHHHHH

UniProtKB: GDNF family receptor alpha-3

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Macromolecule #3: Proto-oncogene tyrosine-protein kinase receptor Ret

MacromoleculeName: Proto-oncogene tyrosine-protein kinase receptor Ret / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.100812 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: LYFSRDAYWE KLYVDQAAGT PLLYVHALRD APEEVPSFRL GQHLYGTYRT RLHENNWICI QEDTGLLYLN RSLDHSSWEK LSVRNHGFP LLTVYLKVFL SPTSLREGEC QWPGCARVYF SFFNTSFPAC SSLKPRELCF PETRPSFRIR ENRPPGTFHQ F RLLPVQFL ...String:
LYFSRDAYWE KLYVDQAAGT PLLYVHALRD APEEVPSFRL GQHLYGTYRT RLHENNWICI QEDTGLLYLN RSLDHSSWEK LSVRNHGFP LLTVYLKVFL SPTSLREGEC QWPGCARVYF SFFNTSFPAC SSLKPRELCF PETRPSFRIR ENRPPGTFHQ F RLLPVQFL CPNISVAYRL LEGEGLPFRC APDSLEVSTR WALDREQREK YELVAVCTVH AGAREEVVMV PFPVTVYDED DS APTFPAG VDTASAVVEF KRKEDTVVAT LRVFDADVVP ASGELVRRYT STLLPGDTWA QQTFRVEHWP NETSVQANGS FVR ATVHDY RLVLNRNLSI SENRTMQLAV LVNDSDFQGP GAGVLLLHFN VSVLPVSLHL PSTYSLSVSR RARRFAQIGK VCVE NCQAF SGINVQYKLH SSGANCSTLG VVTSAEDTSG ILFVNDTKAL RRPKCAELHY MVVATDQQTS RQAQAQLLVT VEGSY VAEE AGCPLSCAVS KRRLECEECG GLGSPTGRCE WRQGDGKGIT RNFSTCSPST KTCPDGHCDV VETQDINICP QDCLRG SIV GGHEPGEPRG IKAGYGTCNC FPEEEKCFCE PEDIQDPLCD ELCRGTHHHH HHHH

UniProtKB: Proto-oncogene tyrosine-protein kinase receptor Ret

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 14 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 8 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration1 mg/mL
BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Average exposure time: 15.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 46729 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: The initial model was generated in RELION.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 114344
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 190
Output model

PDB-6q2s:
Cryo-EM structure of RET/GFRa3/ARTN extracellular complex. The 3D refinement was applied with C2 symmetry.

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