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- EMDB-11777: RET/GDF15/GFRAL extracellular complex negative stain envelope -

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Basic information

Entry
Database: EMDB / ID: EMD-11777
TitleRET/GDF15/GFRAL extracellular complex negative stain envelope
Map data
Sample
  • Complex: Ternary complex of the human RET extracellular domain with the GDF15/GFRAL ligand-co-receptor pair
    • Protein or peptide: Proto-oncogene tyrosine-protein kinase receptor Ret
    • Protein or peptide: GDNF family receptor alpha-like
    • Protein or peptide: Growth/differentiation factor 15
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsAdams SE / Earl CP / 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
DepositionSep 22, 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: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11777.png

Downloads & links

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Map

FileDownload / File: emd_11777.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 3.438 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.07865594 - 0.20507048
Average (Standard dev.)0.00019452676 (±0.010570795)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 412.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.4383.4383.438
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z412.560412.560412.560
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.0790.2050.000

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Supplemental data

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Sample components

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Entire : Ternary complex of the human RET extracellular domain with the GD...

EntireName: Ternary complex of the human RET extracellular domain with the GDF15/GFRAL ligand-co-receptor pair
Components
  • Complex: Ternary complex of the human RET extracellular domain with the GDF15/GFRAL ligand-co-receptor pair
    • Protein or peptide: Proto-oncogene tyrosine-protein kinase receptor Ret
    • Protein or peptide: GDNF family receptor alpha-like
    • Protein or peptide: Growth/differentiation factor 15

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Supramolecule #1: Ternary complex of the human RET extracellular domain with the GD...

SupramoleculeName: Ternary complex of the human RET extracellular domain with the GDF15/GFRAL ligand-co-receptor pair
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Freestyle 293-F / Recombinant plasmid: pCEP
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 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: LYFSRDAYWE KLYVDQAAGT PLLYVHALRD APEEVPSFRL GQHLYGTYRT RLHENNWIRI QEDTGLLYLQ RSLDHSSWEK LSVRNRGFPL LTVYLKVFLS PTSLREGECQ WPGCARVYFS FFNTSFPACS SLKPRELCFP ETRPSFRIRE NRPPGTFHQF RLLPVQFLCP ...String:
LYFSRDAYWE KLYVDQAAGT PLLYVHALRD APEEVPSFRL GQHLYGTYRT RLHENNWIRI QEDTGLLYLQ RSLDHSSWEK LSVRNRGFPL LTVYLKVFLS PTSLREGECQ WPGCARVYFS FFNTSFPACS SLKPRELCFP ETRPSFRIRE NRPPGTFHQF RLLPVQFLCP QISVAYRLLE GEGLPFRSAP DSLEVSTRWA LDREQREKYE LVAVCTVHAG AREEVVMVPF PVTVYDEDDS APTFPAGVDT ASAVVEFKRK EDTVVATLRV FDADVVPASG ELVRRYTSTL LPGDTWAQQT FRVEHWPNET SVQANGSFVR ATVHDYRLVL NRNLSISENR TMQLAVLVND SDFQGPGAGV LLLHFNVSVL PVSLHLPSTY SLSVSRRARR FAQIGKVCVE NCQAFSGINV QYKLHSSGAN CSTLGVVTSA EDTSGILFVN DTKALRRPKC AELHYMVVAT DQQTSRQAQA QLLVTVEGSY VAEEAGCPLS CAVSKRRLEC EECGGLGSPT GRCEWRQGDG KGITRNFSTC SPSTKTCPDG HCDVVETQDI NICPQDCLRG SIVGGHEPGE PRGIKAGYGT CNCFPEEEKC FCEPEDIQDP LCDELCRTGE NLYF

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Macromolecule #2: GDNF family receptor alpha-like

MacromoleculeName: GDNF family receptor alpha-like / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: APLANNCTYL REQCLRDANG CKHAWRVMED ACNDSDPGDP CKMRNSSYCN LSIQYLVESN FQFKECLCTD DFYCTVNKLL GKKCINKSDN VKEDKFKWNL TTRSHHGFKG MWSCLEVAEA CVGDVVCNAQ LASYLKACSA NGNPCDLKQC QAAIRFFYQN IPFNIAQMLA ...String:
APLANNCTYL REQCLRDANG CKHAWRVMED ACNDSDPGDP CKMRNSSYCN LSIQYLVESN FQFKECLCTD DFYCTVNKLL GKKCINKSDN VKEDKFKWNL TTRSHHGFKG MWSCLEVAEA CVGDVVCNAQ LASYLKACSA NGNPCDLKQC QAAIRFFYQN IPFNIAQMLA FCDCAQSDIP CQQSKEALHS KTCAVNMVPP PTCLSVIRSC QNDELCRRHY RTFQSKCWQR VTRKCHEDEN CISTLSKQDL TCSGSDDCKA AYIDILGTVL QVQCTCRTIT QSEESLCKIF QHMLHRKSCF NYPTLSNVKG MALYTRKHAN KITLTGFHSP FNGEAAAHHH HHH

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Macromolecule #3: Growth/differentiation factor 15

MacromoleculeName: Growth/differentiation factor 15 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
APLARARNGD HCPLGPGRCC RLHTVRASLE DLGWADWVLS PREVQVTMCI GACPS QFRA ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCH CI

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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

BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4Shepes
10.0 %C12H22O11sucrose
150.0 mMNaClsodium chloride
1.0 mMCaCl2calcium chloride
100.0 mMC4H11NO3tris

Details: Protein crosslinked by GraFIX method with sucrose/gluteraldehyde gradient
StainingType: NEGATIVE / Material: uranyl acetate
GridModel: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 45 mA

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number grids imaged: 1 / Number real images: 299 / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 42000
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 27551
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 3232
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 5 / Avg.num./class: 1304 / Software - Name: RELION (ver. 2.0)

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Atomic model buiding 1

Initial modelPDB ID:

6q2j

RefinementProtocol: RIGID BODY FIT

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