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- EMDB-27496: Full-length KIT(T417I,delta418-419) dimers -

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Basic information

Entry
Database: EMDB / ID: EMD-27496
TitleFull-length KIT(T417I,delta418-419) dimers
Map dataRelion 3D refinement
Sample
  • Complex: Full-length KIT(T417I,delta418-419) dimers reconstituted in amphipol
    • Protein or peptide: Mast/stem cell growth factor receptor Kit
Keywordsreceptor tyrosine kinase / cell signaling / cancer / cryo-EM / KIT / stem cell factor / oncogenic mutant / extracellular domain / asymmetric interface / structural plasticity / TRANSFERASE
Function / homologyreceptor protein-tyrosine kinase / Isoform 2 of Mast/stem cell growth factor receptor Kit
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.1 Å
AuthorsBertoletti N / Krimmer SG / Mi W / Schlessinger J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention.
Authors: Stefan G Krimmer / Nicole Bertoletti / Yoshihisa Suzuki / Luka Katic / Jyotidarsini Mohanty / Sheng Shu / Sangwon Lee / Irit Lax / Wei Mi / Joseph Schlessinger /
Abstract: The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as ...The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an "Achilles heel" for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.
History
DepositionJul 6, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27496.png

Downloads & links

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Map

FileDownload / File: emd_27496.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRelion 3D refinement
Voxel sizeX=Y=Z: 1.346 Å
Density
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.003418575 - 0.04632148
Average (Standard dev.)0.000012036301 (±0.00077065)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 538.39996 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half1

Fileemd_27496_half_map_1.map
AnnotationHalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half2

Fileemd_27496_half_map_2.map
AnnotationHalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Full-length KIT(T417I,delta418-419) dimers reconstituted in amphipol

EntireName: Full-length KIT(T417I,delta418-419) dimers reconstituted in amphipol
Components
  • Complex: Full-length KIT(T417I,delta418-419) dimers reconstituted in amphipol
    • Protein or peptide: Mast/stem cell growth factor receptor Kit

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Supramolecule #1: Full-length KIT(T417I,delta418-419) dimers reconstituted in amphipol

SupramoleculeName: Full-length KIT(T417I,delta418-419) dimers reconstituted in amphipol
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: Mast/stem cell growth factor receptor Kit

MacromoleculeName: Mast/stem cell growth factor receptor Kit / type: protein_or_peptide / ID: 1
Details: MTILCWLALLSTLTAVNA signal peptide NADYKDDDDKRPHAM expression tag (NAG) post-translational glycosylation Mutation T417I Deletion mutation Y418,D419 Mutation K623A GSHHHHHH expression tag
Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTILCWLALL STLTAVNADY KDDDDKRPHA MGEPSPPSIH PGKSDLIVRV GDEIRLLCTD PGFVKWTFEI LDETNENKQN EWITEKAEAT NTGKYTCTNK HGLSNSIYVF VRDPAKLFLV DRSLYGKEDN DTLVRCPLTD PEVTNYSLKG CQGKPLPKDL RFIPDPKAGI ...String:
MTILCWLALL STLTAVNADY KDDDDKRPHA MGEPSPPSIH PGKSDLIVRV GDEIRLLCTD PGFVKWTFEI LDETNENKQN EWITEKAEAT NTGKYTCTNK HGLSNSIYVF VRDPAKLFLV DRSLYGKEDN DTLVRCPLTD PEVTNYSLKG CQGKPLPKDL RFIPDPKAGI MIKSVKRAYH RLCLHCSVDQ EGKSVLSEKF ILKVRPAFKA VPVVSVSKAS YLLREGEEFT VTCTIKDVSS SVYSTWKREN SQTKLQEKYN SWHHGDFNYE RQATLTISSA RVNDSGVFMC YANNTFGSAN VTTTLEVVDK GFINIFPMIN TTVFVNDGEN VDLIVEYEAF PKPEHQQWIY MNRTFTDKWE DYPKSENESN IRYVSELHLT RLKGTEGGTY TFLVSNSDVN AAIAFNVYVN TKPEILIRLV NGMLQCVAAG FPEPTIDWYF CPGTEQRCSA SVLPVDVQTL NSSGPPFGKL VVQSSIDSSA FKHNGTVECK AYNDVGKTSA YFNFAFKEQI HPHTLFTPLL IGFVIVAGMM CIIVMILTYK YLQKPMYEVQ WKVVEEINGN NYVYIDPTQL PYDHKWEFPR NRLSFGKTLG AGAFGKVVEA TAYGLIKSDA AMTVAVAMLK PSAHLTEREA LMSELKVLSY LGNHMNIVNL LGACTIGGPT LVITEYCCYG DLLNFLRRKR DSFICSKQED HAEAALYKNL LHSKESSCSD STNEYMDMKP GVSYVVPTKA DKRRSVRIGS YIERDVTPAI MEDDELALDL EDLLSFSYQV AKGMAFLASK NCIHRDLAAR NILLTHGRIT KICDFGLARD IKNDSNYVVK GNARLPVKWM APESIFNCVY TFESDVWSYG IFLWELFSLG SSPYPGMPVD SKFYKMIKEG FRMLSPEHAP AEMYDIMKTC WDADPLKRPT FKQIVQLIEK QISESTNHIY SNLANCSPNR QKPVVDHSVR INSVGSTASS SQPLLVHDDV GSHHHHHH

UniProtKB: Isoform 2 of Mast/stem cell growth factor receptor Kit

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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

Concentration5.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
200.0 mMNaClSodium chlorideSodium chloride
0.1 %C13H17F13NO4P(1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Blotting time 3 sec, blotting force 0..
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DetailsSerialEM COMA-FREE ALIGNMENT
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8397 / Average exposure time: 11.134 sec. / Average electron dose: 64.76 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2457127
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 13.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 213615
FSC plot (resolution estimation)

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