+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2648 | |||||||||
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Title | stem cell factor-induced intact KIT dimers | |||||||||
Map data | Reconstruction of intact, kinase-dead KIT-SCF complex | |||||||||
Sample |
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Keywords | KIT / c-Kit / SCF / stem cell factor / transmembrane / receptor tyrosine kinase / RTK | |||||||||
Function / homology | : Function and homology information | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 28.8 Å | |||||||||
Authors | Opatowsky Y / Bleichert F / Unger VM / Schlessinger J | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2014 Title: Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers. Authors: Yarden Opatowsky / Irit Lax / Francisco Tomé / Franziska Bleichert / Vinzenz M Unger / Joseph Schlessinger / Abstract: Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form ...Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions in the membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions occurring during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2648.map.gz | 971.5 KB | EMDB map data format | |
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Header (meta data) | emd-2648-v30.xml emd-2648.xml | 11 KB 11 KB | Display Display | EMDB header |
Images | emd_2648.png | 501.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2648 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2648 | HTTPS FTP |
-Validation report
Summary document | emd_2648_validation.pdf.gz | 198.7 KB | Display | EMDB validaton report |
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Full document | emd_2648_full_validation.pdf.gz | 197.8 KB | Display | |
Data in XML | emd_2648_validation.xml.gz | 4.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2648 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2648 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2648.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of intact, kinase-dead KIT-SCF complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 5.5 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Intact human KIT dimer in complex with stem cell factor (SCF)dimer
Entire | Name: Intact human KIT dimer in complex with stem cell factor (SCF)dimer |
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Components |
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-Supramolecule #1000: Intact human KIT dimer in complex with stem cell factor (SCF)dimer
Supramolecule | Name: Intact human KIT dimer in complex with stem cell factor (SCF)dimer type: sample / ID: 1000 / Oligomeric state: one dimer of SCF binds to KIT dimer / Number unique components: 2 |
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Molecular weight | Theoretical: 290 KDa / Method: SDS-PAGE |
-Macromolecule #1: stem cell growth factor
Macromolecule | Name: stem cell growth factor / type: protein_or_peptide / ID: 1 / Name.synonym: SCF / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane |
Molecular weight | Theoretical: 15 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET11 |
Sequence | UniProtKB: UNIPROTKB: P21583 |
-Macromolecule #2: KIT
Macromolecule | Name: KIT / type: protein_or_peptide / ID: 2 / Oligomeric state: dimer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET11 |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.05 mg/mL |
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Buffer | pH: 7.4 / Details: 200mM NaCl, 20mM HEPES,15% glycerol, 0.05% DDM |
Staining | Type: NEGATIVE Details: grids with adsorbed protein were floated on 3 drops of 1% uranyl formate for 30 seconds each |
Grid | Details: 400 mesh copper grid with continuous thin carbon support over holy carbon film |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI 12 |
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Date | Nov 27, 2008 |
Image recording | Category: CCD / Film or detector model: OTHER / Number real images: 400 / Average electron dose: 20 e/Å2 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 30000 |
Sample stage | Specimen holder model: OTHER / Tilt angle max: 55 |
-Image processing
Details | Particles of tilt pairs (at 0 and 55 degrees)were manually selected using WEB. Initial reference-free alignment and classification of the untilted views was performed using IMAGIC-5. A representative class average was used for reference-based alignment, followed by cycles of multireference alignment and classification. Three out of 8 classes were used in random conical tilt reconstruction using SPIDER. |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 28.8 Å / Resolution method: OTHER / Software - Name: SPIDER / Number images used: 1900 |
Final two d classification | Number classes: 3 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: Chimera |
Details | The domains were separately fitted by manual docking using Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: Chimera |
Details | The domains were separately fitted by manual docking using Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |