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- EMDB-13688: Negative stain EM map of the extracellular domain of the RET(C634... -

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Basic information

Entry
Database: EMDB / ID: EMD-13688
TitleNegative stain EM map of the extracellular domain of the RET(C634R) mutant dimer
Map dataNegative stain EM map of RET(C634R) dimer
Sample
  • Complex: RET(C634R) mutant dimer
KeywordsRET / Dimer / RTK / Oncogenesis / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 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_13688.png

Downloads & links

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Map

FileDownload / File: emd_13688.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM map of RET(C634R) dimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.25 Å/pix.
x 180 pix.
= 405. Å		2.25 Å/pix.
x 180 pix.
= 405. Å		2.25 Å/pix.
x 180 pix.
= 405. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.25 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-0.97357 - 5.3001294
Average (Standard dev.)0.0060853823 (±0.11886327)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 405.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13688_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) mutant dimer

EntireName: RET(C634R) mutant dimer
Components
  • Complex: RET(C634R) mutant dimer

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Supramolecule #1: RET(C634R) mutant dimer

SupramoleculeName: RET(C634R) mutant dimer / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 185 KDa

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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

Concentration0.005 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: 40 / 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: 3490 / Details: Manual particle picking
Startup modelType of model: OTHER
Details: The initial model was built based on 2D class images in EMAN2.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.3) / Number images used: 3490
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2
Final angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2
FSC plot (resolution estimation)

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