+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27410 | |||||||||
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Title | Ectodomain of full-length KIT(DupA502,Y503)-SCF dimers | |||||||||
Map data | Ectodomain local refinement of KIT(DupA502,Y503)-SCF dimers | |||||||||
Sample |
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Function / homology | : / : Function and homology information | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.17 Å | |||||||||
Authors | Bertoletti N / Krimmer SG / Mi W / Schlessinger J | |||||||||
Funding support | 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27410.map.gz | 175.4 MB | EMDB map data format | |
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Header (meta data) | emd-27410-v30.xml emd-27410.xml | 22.5 KB 22.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27410_fsc.xml | 15.6 KB | Display | FSC data file |
Images | emd_27410.png | 59.9 KB | ||
Masks | emd_27410_msk_1.map | 347.6 MB | Mask map | |
Others | emd_27410_additional_1.map.gz emd_27410_half_map_1.map.gz emd_27410_half_map_2.map.gz | 328.2 MB 322.6 MB 322.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27410 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27410 | HTTPS FTP |
-Validation report
Summary document | emd_27410_validation.pdf.gz | 742.2 KB | Display | EMDB validaton report |
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Full document | emd_27410_full_validation.pdf.gz | 741.7 KB | Display | |
Data in XML | emd_27410_validation.xml.gz | 23.9 KB | Display | |
Data in CIF | emd_27410_validation.cif.gz | 31.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27410 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27410 | HTTPS FTP |
-Related structure data
Related structure data | 8dfpMC 8dfmC 8dfqC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_27410.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Ectodomain local refinement of KIT(DupA502,Y503)-SCF dimers | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.346 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_27410_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Sharpened main map
File | emd_27410_additional_1.map | ||||||||||||
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Annotation | Sharpened main map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map B of main map
File | emd_27410_half_map_1.map | ||||||||||||
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Annotation | Half map B of main map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map A of main map
File | emd_27410_half_map_2.map | ||||||||||||
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Annotation | Half map A of main map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Full-length KIT(DupA502,Y503)-SCF dimers reconstituted in amphipol
Entire | Name: Full-length KIT(DupA502,Y503)-SCF dimers reconstituted in amphipol |
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Components |
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-Supramolecule #1: Full-length KIT(DupA502,Y503)-SCF dimers reconstituted in amphipol
Supramolecule | Name: Full-length KIT(DupA502,Y503)-SCF dimers reconstituted in amphipol type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Homo sapiens (human) / Location in cell: membrane |
Molecular weight | Theoretical: 250 KDa |
-Macromolecule #1: Isoform 2 of Mast/stem cell growth factor receptor Kit
Macromolecule | Name: Isoform 2 of Mast/stem cell growth factor receptor Kit type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 110.891258 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: MTILCWLALL STLTAVNADY KDDDDKRPHA MGEPSPPSIH PGKSDLIVRV GDEIRLLCTD PGFVKWTFEI LDETNENKQN EWITEKAEA TNTGKYTCTN KHGLSNSIYV FVRDPAKLFL VDRSLYGKED NDTLVRCPLT DPEVTNYSLK GCQGKPLPKD L RFIPDPKA ...String: MTILCWLALL STLTAVNADY KDDDDKRPHA MGEPSPPSIH PGKSDLIVRV GDEIRLLCTD PGFVKWTFEI LDETNENKQN EWITEKAEA TNTGKYTCTN KHGLSNSIYV FVRDPAKLFL VDRSLYGKED NDTLVRCPLT DPEVTNYSLK GCQGKPLPKD L RFIPDPKA GIMIKSVKRA YHRLCLHCSV DQEGKSVLSE KFILKVRPAF KAVPVVSVSK ASYLLREGEE FTVTCTIKDV SS SVYSTWK RENSQTKLQE KYNSWHHGDF NYERQATLTI SSARVNDSGV FMCYANNTFG SANVTTTLEV VDKGFINIFP MIN TTVFVN DGENVDLIVE YEAFPKPEHQ QWIYMNRTFT DKWEDYPKSE NESNIRYVSE LHLTRLKGTE GGTYTFLVSN SDVN AAIAF NVYVNTKPEI LTYDRLVNGM LQCVAAGFPE PTIDWYFCPG TEQRCSASVL PVDVQTLNSS GPPFGKLVVQ SSIDS SAFK HNGTVECKAY NDVGKTSAYA YFNFAFKEQI HPHTLFTPLL IGFVIVAGMM CIIVMILTYK YLQKPMYEVQ WKVVEE ING NNYVYIDPTQ LPYDHKWEFP RNRLSFGKTL GAGAFGKVVE ATAYGLIKSD AAMTVAVAML KPSAHLTERE ALMSELK VL SYLGNHMNIV NLLGACTIGG PTLVITEYCC YGDLLNFLRR KRDSFICSKQ EDHAEAALYK NLLHSKESSC SDSTNEYM D MKPGVSYVVP TKADKRRSVR IGSYIERDVT PAIMEDDELA LDLEDLLSFS YQVAKGMAFL ASKNCIHRDL AARNILLTH GRITKICDFG LARDIKNDSN YVVKGNARLP VKWMAPESIF NCVYTFESDV WSYGIFLWEL FSLGSSPYPG MPVDSKFYKM IKEGFRMLS PEHAPAEMYD IMKTCWDADP LKRPTFKQIV QLIEKQISES TNHIYSNLAN CSPNRQKPVV DHSVRINSVG S TASSSQPL LVHDDVGSHH HHHH |
-Macromolecule #2: Soluble KIT ligand
Macromolecule | Name: Soluble KIT ligand / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 14.742981 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: ICRNRVTNNV KDVTKLVANL PKDYMITLKY VPGMDVLPSH CWISEMVVQL SDSLTDLLDK FSNISEGLSN YSIIDKLVNI VDDLVECVK ENSSKDLKKS FKSPEPRLFT PEEFFRIFNR SIDAFKDFV |
-Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 2 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 5.6 mg/mL | ||||||||||||
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Buffer | pH: 7.4 Component:
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Grid | Model: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Blotting time 3 sec, blotting force 0.. | ||||||||||||
Details | This sample was monodisperse. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Details | SerialEM COMA-FREE ALIGNMENT |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8456 / Average exposure time: 11.134 sec. / Average electron dose: 64.52 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 64000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |