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- EMDB-2648: stem cell factor-induced intact KIT dimers -

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Basic information

Entry
Database: EMDB / ID: EMD-2648
Titlestem cell factor-induced intact KIT dimers
Map dataReconstruction of intact, kinase-dead KIT-SCF complex
Sample
  • Sample: Intact human KIT dimer in complex with stem cell factor (SCF)dimer
  • Protein or peptide: stem cell growth factor
  • Protein or peptide: KIT
KeywordsKIT / c-Kit / SCF / stem cell factor / transmembrane / receptor tyrosine kinase / RTK
Function / homology
Function and homology information


positive regulation of myeloid leukocyte differentiation / stem cell factor receptor binding / mast cell migration / positive regulation of hematopoietic stem cell proliferation / positive regulation of hematopoietic progenitor cell differentiation / negative regulation of mast cell apoptotic process / melanocyte migration / positive regulation of melanocyte differentiation / myeloid leukocyte differentiation / positive regulation of mast cell proliferation ...positive regulation of myeloid leukocyte differentiation / stem cell factor receptor binding / mast cell migration / positive regulation of hematopoietic stem cell proliferation / positive regulation of hematopoietic progenitor cell differentiation / negative regulation of mast cell apoptotic process / melanocyte migration / positive regulation of melanocyte differentiation / myeloid leukocyte differentiation / positive regulation of mast cell proliferation / mast cell apoptotic process / mast cell proliferation / positive regulation of Ras protein signal transduction / positive regulation of leukocyte migration / neural crest cell migration / embryonic hemopoiesis / Regulation of KIT signaling / ectopic germ cell programmed cell death / hematopoietic progenitor cell differentiation / T cell proliferation / ovarian follicle development / positive regulation of T cell proliferation / extrinsic apoptotic signaling pathway in absence of ligand / Transcriptional and post-translational regulation of MITF-M expression and activity / cytokine activity / filopodium / growth factor activity / Signaling by SCF-KIT / male gonad development / Constitutive Signaling by Aberrant PI3K in Cancer / PIP3 activates AKT signaling / lamellipodium / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / RAF/MAP kinase cascade / Ras protein signal transduction / cytoskeleton / cell adhesion / positive regulation of cell population proliferation / extracellular space / extracellular region / plasma membrane / cytoplasm
Similarity search - Function
Stem cell factor / Stem cell factor / Four-helical cytokine-like, core
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 28.8 Å
AuthorsOpatowsky Y / Bleichert F / Unger VM / Schlessinger J
CitationJournal: 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
DepositionMay 14, 2014-
Header (metadata) releaseJun 4, 2014-
Map releaseJun 4, 2014-
UpdateJun 4, 2014-
Current statusJun 4, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2648.png

Downloads & links

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Map

FileDownload / File: emd_2648.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of intact, kinase-dead KIT-SCF complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.5 Å/pix.
x 64 pix.
= 352. Å		5.5 Å/pix.
x 64 pix.
= 352. Å		5.5 Å/pix.
x 64 pix.
= 352. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.4 / Movie #1: 2.4
Minimum - Maximum-4.12396765 - 12.35673618
Average (Standard dev.)0.00694332 (±0.42488474)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.55.55.5
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z352.000352.000352.000
α/β/γ90.00090.00090.000
start NX/NY/NZ0-51-100
NX/NY/NZ82103201
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-4.12412.3570.007

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Supplemental data

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Sample components

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Entire : Intact human KIT dimer in complex with stem cell factor (SCF)dimer

EntireName: Intact human KIT dimer in complex with stem cell factor (SCF)dimer
Components
  • Sample: Intact human KIT dimer in complex with stem cell factor (SCF)dimer
  • Protein or peptide: stem cell growth factor
  • Protein or peptide: KIT

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Supramolecule #1000: Intact human KIT dimer in complex with stem cell factor (SCF)dimer

SupramoleculeName: 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
Molecular weightTheoretical: 290 KDa / Method: SDS-PAGE

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Macromolecule #1: stem cell growth factor

MacromoleculeName: stem cell growth factor / type: protein_or_peptide / ID: 1 / Name.synonym: SCF / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane
Molecular weightTheoretical: 15 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET11
SequenceUniProtKB: Kit ligand

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Macromolecule #2: KIT

MacromoleculeName: KIT / type: protein_or_peptide / ID: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET11

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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

Concentration0.05 mg/mL
BufferpH: 7.4 / Details: 200mM NaCl, 20mM HEPES,15% glycerol, 0.05% DDM
StainingType: NEGATIVE
Details: grids with adsorbed protein were floated on 3 drops of 1% uranyl formate for 30 seconds each
GridDetails: 400 mesh copper grid with continuous thin carbon support over holy carbon film
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
DateNov 27, 2008
Image recordingCategory: CCD / Film or detector model: OTHER / Number real images: 400 / Average electron dose: 20 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 30000
Sample stageSpecimen holder model: OTHER / Tilt angle max: 55

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Image processing

DetailsParticles 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.
Final reconstructionApplied 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 classificationNumber classes: 3

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Atomic model buiding 1

Initial modelPDB ID:

2e9w

SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

1t45

SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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