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- EMDB-13421: Cryo-EM maps of the RET/GDF15/GFRAL complex -

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Basic information

Entry
Database: EMDB / ID: EMD-13421
TitleCryo-EM maps of the RET/GDF15/GFRAL complex
Map dataPost-processed masked map. Map has been z-flipped to match the orientation of the published structure of the RET/GDF15/GRFAL complex (PDB ID 6Q2J).
Sample
  • Complex: The extracellular domain complex of RET/GDF15/GFRAL
    • Complex: The extracellular domain of proto-oncogene tyrosine-protein kinase receptor Ret
    • Complex: The mature growth and differentiation factor 15 with N-terminal Fc tags.
    • Complex: The extracellular domain of GDNF family receptor alpha-like
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsLiu YL / Muench SP / Goldman A
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Academy of Finland322609 United Kingdom
Academy of Finland286429 United Kingdom
Other private10661 United Kingdom
European Molecular Biology Organization (EMBO)8476 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M021610/1 United Kingdom
CitationJournal: J Biol Chem / Year: 2022
Title: Unexpected structures formed by the kinase RET C634R mutant extracellular domain suggest potential oncogenic mechanisms in MEN2A.
Authors: Yixin Liu / Orquidea De Castro Ribeiro / Outi Haapanen / Gregory B Craven / Vivek Sharma / Stephen P Muench / Adrian Goldman /
Abstract: The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. ...The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. Mutations that disrupt the disulfide network in the extracellular domain (ECD) of RET drive multiple endocrine neoplasia type 2A (MEN2A), a hereditary syndrome associated with the development of thyroid cancers. However, structural details of how specific mutations affect RET are unclear. Here, we present the first structural insights into the ECD of the RET(C634R) mutant, the most common mutation in MEN2A. Using electron microscopy, we demonstrate that the C634R mutation causes ligand-independent dimerization of the RET ECD, revealing an unusual tail-to-tail conformation that is distinct from the ligand-induced signaling dimer of WT RET. Additionally, we show that the RET ECD dimer can form complexes with at least two of the canonical RET ligands and that these complexes form very different structures than WT RET ECD upon ligand binding. In conclusion, this structural analysis of cysteine-mutant RET ECD suggests a potential key mechanism of cancer induction in MEN2A, both in the absence and presence of its native ligands, and may offer new targets for therapeutic intervention.
History
DepositionAug 17, 2021-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateSep 28, 2022-
Current statusSep 28, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13421.png

Downloads & links

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Map

FileDownload / File: emd_13421.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed masked map. Map has been z-flipped to match the orientation of the published structure of the RET/GDF15/GRFAL complex (PDB ID 6Q2J).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.13 Å/pix.
x 128 pix.
= 272.64 Å		2.13 Å/pix.
x 128 pix.
= 272.64 Å		2.13 Å/pix.
x 128 pix.
= 272.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.13 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055
Minimum - Maximum-0.19524585 - 0.3401795
Average (Standard dev.)0.0012032468 (±0.012794067)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 272.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13421_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Different conformations of the RET/GDF15/GFRAL complex (Class 2)

Fileemd_13421_additional_1.map
AnnotationDifferent conformations of the RET/GDF15/GFRAL complex (Class 2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Different conformations of the RET/GDF15/GFRAL complex (Class 1)

Fileemd_13421_additional_2.map
AnnotationDifferent conformations of the RET/GDF15/GFRAL complex (Class 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Different conformations of the RET/GDF15/GFRAL complex (Class 3)

Fileemd_13421_additional_3.map
AnnotationDifferent conformations of the RET/GDF15/GFRAL complex (Class 3)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map2, z-flipped

Fileemd_13421_half_map_1.map
AnnotationHalf map2, z-flipped
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map1, z-flipped

Fileemd_13421_half_map_2.map
AnnotationHalf map1, z-flipped
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The extracellular domain complex of RET/GDF15/GFRAL

EntireName: The extracellular domain complex of RET/GDF15/GFRAL
Components
  • Complex: The extracellular domain complex of RET/GDF15/GFRAL
    • Complex: The extracellular domain of proto-oncogene tyrosine-protein kinase receptor Ret
    • Complex: The mature growth and differentiation factor 15 with N-terminal Fc tags.
    • Complex: The extracellular domain of GDNF family receptor alpha-like

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Supramolecule #1: The extracellular domain complex of RET/GDF15/GFRAL

SupramoleculeName: The extracellular domain complex of RET/GDF15/GFRAL / type: complex / ID: 1 / Parent: 0
Details: The extracellular domain of the proto-oncogene tyrosine-protein kinase receptor Ret. Mature growth and differentiation factor 15 with two N-terminal Fc tags. The extracellular domain of GDNF ...Details: The extracellular domain of the proto-oncogene tyrosine-protein kinase receptor Ret. Mature growth and differentiation factor 15 with two N-terminal Fc tags. The extracellular domain of GDNF family receptor alpha-like.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 40 KDa

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Supramolecule #2: The extracellular domain of proto-oncogene tyrosine-protein kinas...

SupramoleculeName: The extracellular domain of proto-oncogene tyrosine-protein kinase receptor Ret
type: complex / ID: 2 / Parent: 1
Details: RET is secreted and purified from cell culture medium.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: The mature growth and differentiation factor 15 with N-terminal F...

SupramoleculeName: The mature growth and differentiation factor 15 with N-terminal Fc tags.
type: complex / ID: 3 / Parent: 1
Details: Fc-GDF15 was secreted and purified from cell culture medium using protein A resin.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #4: The extracellular domain of GDNF family receptor alpha-like

SupramoleculeName: The extracellular domain of GDNF family receptor alpha-like
type: complex / ID: 4 / Parent: 1
Details: GFRAL is secreted and purified from cell culture medium using Strep-Tactin affinity purification.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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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: 8
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
100.0 mMNaClsodium chloride
1.0 mMCaCl2calcium chloride

Details: Buffer was prepared fresh and filter steriled.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 6 second before plunging.
DetailsThis sample was >90% pure.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Average exposure time: 1.5 sec. / Average electron dose: 60.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 75000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1135874 / Details: auto-picked using crYOLO
Startup modelType of model: OTHER / Details: Initial model built in Relion
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 142083
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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