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- EMDB-13689: Negative stain EM map of the extracellular domain of the RET(C634... -

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Basic information

Entry
Database: EMDB / ID: EMD-13689
TitleNegative stain EM map of the extracellular domain of the RET(C634R)/GDF15/GFRAL complex
Map dataNegative stain EM map of the extracellular domain of the RET(C634R)/GDF15/GFRAL complex. The map has been z-flipped to match the structure of the wild-type complex (PDB ID 6Q2J).
Sample
  • Complex: RET(C634R)/GDF15/GFRAL
    • Complex: RET(C634R) dimer
    • Complex: GDF15 dimer
    • Complex: GFRAL
KeywordsRET / Complex / RTK / Oncogenesis / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsLiu Y / Muench SP / Goldman A
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Academy of Finland322609 United Kingdom
Academy of Finland286429 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
DepositionOct 7, 2021-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13689.png

Downloads & links

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Map

FileDownload / File: emd_13689.map.gz / Format: CCP4 / Size: 2.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM map of the extracellular domain of the RET(C634R)/GDF15/GFRAL complex. The map has been z-flipped to match the structure of the wild-type complex (PDB ID 6Q2J).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.25 Å/pix.
x 84 pix.
= 441. Å		5.25 Å/pix.
x 84 pix.
= 441. Å		5.25 Å/pix.
x 84 pix.
= 441. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.25 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5
Minimum - Maximum-13.391591 - 22.847235000000001
Average (Standard dev.)0.028749669 (±0.576122)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions848484
Spacing848484
CellA=B=C: 441.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13689_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RET(C634R)/GDF15/GFRAL

EntireName: RET(C634R)/GDF15/GFRAL
Components
  • Complex: RET(C634R)/GDF15/GFRAL
    • Complex: RET(C634R) dimer
    • Complex: GDF15 dimer
    • Complex: GFRAL

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Supramolecule #1: RET(C634R)/GDF15/GFRAL

SupramoleculeName: RET(C634R)/GDF15/GFRAL / type: complex / ID: 1 / Parent: 0
Details: The extracellular domain complex was reconstituted using individually purified protein components and purified via gel filtration.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: RET(C634R) dimer

SupramoleculeName: RET(C634R) dimer / type: complex / ID: 2 / Parent: 1
Details: RET(C634R)mutant dimer generated by affinity purification and gel filtration
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: GDF15 dimer

SupramoleculeName: GDF15 dimer / type: complex / ID: 3 / Parent: 1
Details: GDF15 dimer was originally expressed as an Fc-tagged fusion and purified via affinity purification. Tag was cleaved after complex assembly.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: GFRAL

SupramoleculeName: GFRAL / type: complex / ID: 4 / Parent: 1
Details: The extracellular domain of GFRAL was purified via affinity purification and gel filtration.
Source (natural)Organism: Homo sapiens (human)

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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

Concentration0.01 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
100.0 mMNaClsodium chloride
1.0 mMCaCl2calcium chloride

Details: Buffer was made fresh and filtered through a 0.2 um filter.
StainingType: NEGATIVE / Material: Uranyl Acetate / Details: 2% Uranyl acetate solution
GridMaterial: COPPER / Mesh: 300
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TECNAI 20
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 1 / Number real images: 68 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 50000

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Image processing

Particle selectionNumber selected: 11278 / Details: Manual particle picking
Startup modelType of model: OTHER
Details: The initial model was built based on 2D class images in EMAN2.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.3) / Details: Particles were low pass filtered to 20A. / Number images used: 3908
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2
Final angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2
Final 3D classificationNumber classes: 3 / Avg.num./class: 3500 / Software - Name: EMAN2 (ver. 2.3)
Details: Multi-refinement using three initial models built in EMAN2 as starting models.
FSC plot (resolution estimation)

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