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- EMDB-20708: B-Raf:14-3-3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20708
TitleB-Raf:14-3-3 complex
Map data
Sample
  • Complex: Ternary complex of B-Raf kinase domain dimer and 14-3-3 dimer
    • Protein or peptide: 14-3-3 zeta
    • Protein or peptide: Serine/threonine-protein kinase B-raf
Function / homology
Function and homology information


trehalose metabolism in response to stress / CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / head morphogenesis / Signalling to p38 via RIT and RIN / myeloid progenitor cell differentiation / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling ...trehalose metabolism in response to stress / CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / head morphogenesis / Signalling to p38 via RIT and RIN / myeloid progenitor cell differentiation / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / endothelial cell apoptotic process / negative regulation of fibroblast migration / positive regulation of glucose transmembrane transport / establishment of protein localization to membrane / mitogen-activated protein kinase kinase binding / regulation of T cell differentiation / Negative feedback regulation of MAPK pathway / positive regulation of axonogenesis / Frs2-mediated activation / stress fiber assembly / positive regulation of axon regeneration / face development / synaptic vesicle exocytosis / somatic stem cell population maintenance / MAP kinase kinase activity / thyroid gland development / MAP kinase kinase kinase activity / negative regulation of endothelial cell apoptotic process / positive regulation of substrate adhesion-dependent cell spreading / response to cAMP / positive regulation of stress fiber assembly / ERK1 and ERK2 cascade / cellular response to calcium ion / substrate adhesion-dependent cell spreading / cellular response to nerve growth factor stimulus / thymus development / long-term synaptic potentiation / animal organ morphogenesis / Spry regulation of FGF signaling / RAF activation / Signaling by high-kinase activity BRAF mutants / visual learning / MAP2K and MAPK activation / epidermal growth factor receptor signaling pathway / response to peptide hormone / Negative regulation of MAPK pathway / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / MAPK cascade / Signaling by BRAF and RAF1 fusions / cellular response to xenobiotic stimulus / presynapse / cell body / positive regulation of peptidyl-serine phosphorylation / T cell differentiation in thymus / regulation of cell population proliferation / T cell receptor signaling pathway / scaffold protein binding / negative regulation of neuron apoptotic process / positive regulation of ERK1 and ERK2 cascade / non-specific serine/threonine protein kinase / neuron projection / protein kinase activity / protein phosphorylation / protein serine kinase activity / intracellular membrane-bounded organelle / protein serine/threonine kinase activity / calcium ion binding / protein-containing complex binding / positive regulation of gene expression / negative regulation of apoptotic process / mitochondrion / ATP binding / identical protein binding / nucleus / plasma membrane / cytosol
Similarity search - Function
Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain ...Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C1-like domain superfamily / 14-3-3 proteins signature 2. / 14-3-3 protein, conserved site / 14-3-3 proteins signature 1. / 14-3-3 protein / 14-3-3 homologues / 14-3-3 domain / 14-3-3 domain superfamily / 14-3-3 protein / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Ubiquitin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
14-3-3 zeta / Serine/threonine-protein kinase B-raf
Similarity search - Component
Biological speciesHomo sapiens (human) / Asian cotton leafworm (butterflies/moths)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKondo Y / Ognjenovic J / Banerjee S / Karandur D / Merk A / Kulhanek K / Wong K / Roose JP / Subramaniam S / Kuriyan J
Funding support United States, Canada, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P01-AI091580 United States
Canada Excellence Research Chair Award Canada
Citation
Journal: Science / Year: 2019
Title: Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases.
Authors: Yasushi Kondo / Jana Ognjenović / Saikat Banerjee / Deepti Karandur / Alan Merk / Kayla Kulhanek / Kathryn Wong / Jeroen P Roose / Sriram Subramaniam / John Kuriyan /
Abstract: Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo-electron microscopy structural analysis of a phosphorylated B-Raf kinase ...Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo-electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of ~3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical "active" conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.
#1: Journal: Science / Year: 2019
Title: Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases
Authors: Kondo Y / Ognjenovic J / Banerjee S / Karandur D / Merk A / Kulhanek K / Wong K / Roose JP / Subramaniam S / Kuriyan J
History
DepositionSep 11, 2019-
Header (metadata) releaseSep 25, 2019-
Map releaseSep 25, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uan
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20708.png

Downloads & links

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Map

FileDownload / File: emd_20708.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.056520123 - 0.10374748
Average (Standard dev.)-0.00000212983 (±0.0037005271)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.000212.000212.000
α/β/γ90.00090.00090.000
start NX/NY/NZ1548552
NX/NY/NZ198170217
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0570.104-0.000

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Supplemental data

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Sample components

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Entire : Ternary complex of B-Raf kinase domain dimer and 14-3-3 dimer

EntireName: Ternary complex of B-Raf kinase domain dimer and 14-3-3 dimer
Components
  • Complex: Ternary complex of B-Raf kinase domain dimer and 14-3-3 dimer
    • Protein or peptide: 14-3-3 zeta
    • Protein or peptide: Serine/threonine-protein kinase B-raf

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Supramolecule #1: Ternary complex of B-Raf kinase domain dimer and 14-3-3 dimer

SupramoleculeName: Ternary complex of B-Raf kinase domain dimer and 14-3-3 dimer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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Macromolecule #1: 14-3-3 zeta

MacromoleculeName: 14-3-3 zeta / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Asian cotton leafworm (butterflies/moths)
Molecular weightTheoretical: 28.108514 KDa
SequenceString: MSVDKEELVQ RAKLAEQAER YDDMAAAMKE VTETGVELSN EERNLLSVAY KNVVGARRSS WRVISSIEQK TEGSERKQQM AKEYRVKVE KELREICYDV LGLLDKHLIP KASNPESKVF YLKMKGDYYR YLAEVATGET RNSVVEDSQK AYQDAFEISK A KMQPTHPI ...String:
MSVDKEELVQ RAKLAEQAER YDDMAAAMKE VTETGVELSN EERNLLSVAY KNVVGARRSS WRVISSIEQK TEGSERKQQM AKEYRVKVE KELREICYDV LGLLDKHLIP KASNPESKVF YLKMKGDYYR YLAEVATGET RNSVVEDSQK AYQDAFEISK A KMQPTHPI RLGLALNFSV FYYEILNSPD KACQLAKQAF DDAIAELDTL NEDSYKDSTL IMQLLRDNLT LWTSDTQGDG DE PAEGGDN

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Macromolecule #2: Serine/threonine-protein kinase B-raf

MacromoleculeName: Serine/threonine-protein kinase B-raf / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 84.781922 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: SGMAALSGGG GGGAEPGQAL FNGDMEPEAG AGAGAAASSA ADPAIPEEVW NIKQMIKLTQ EHIEALLDKF GGEHNPPSIY LEAYEEYTS KLDALQQREQ QLLESLGNGT DFSVSSSASM DTVTSSSSSS LSVLPSSLSV FQNPTDVARS NPKSPQKPIV R VFLPNKQR ...String:
SGMAALSGGG GGGAEPGQAL FNGDMEPEAG AGAGAAASSA ADPAIPEEVW NIKQMIKLTQ EHIEALLDKF GGEHNPPSIY LEAYEEYTS KLDALQQREQ QLLESLGNGT DFSVSSSASM DTVTSSSSSS LSVLPSSLSV FQNPTDVARS NPKSPQKPIV R VFLPNKQR TVVPARCGVT VRDSLKKALM MRGLIPECCA VYRIQDGEKK PIGWDTDISW LTGEELHVEV LENVPLTTHN FV RKTFFTL AFCDFCRKLL FQGFRCQTCG YKFHQRCSTE VPLMCVNYDQ LDLLFVSKFF EHHPIPQEEA SLAETALTSG SSP SAPASD SIGPQILTSP SPSKSIPIPQ PFRPADEDHR NQFGQRDRSS SAPNVHINCM EPVNIDDLIR DQGFRGDGGS TTGL SATPP ASLPGSLTNV KALQKSPGPQ RERKSSSSSE DRNRMKTLGR RDSSDDWEIP DGQITVGQRI GSGSFGTVYK GKWHG DVAV KMLNVTAPTP QQLQAFKNEV GVLRKTRHVN ILLFMGYSTK PQLAIVTQWC EGSSLYHHLH IIETKFEMIK LIDIAR QTA QGMDYLHAKS IIHRDLKSNN IFLHEDLTVK IGDFGLATVK SRWSGSHQFE QLSGSILWMA PEVIRMQDKN PYSFQSD VY AFGIVLYELM TGQLPYSNIN NRDQIIFMVG RGYLSPDLSK VRSNCPKAMK RLMAECLKKK RDERPLFPQI LASIELLA R SLPKIHRSA(SEP) EPSLNRAGFQ TEDFSLYACA SPKTPIQAGG YGAFPVH

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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

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMTris-HClTris
150.0 mMsodium chloride
0.5 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 278 K / Instrument: LEICA EM GP

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 113313
FSC plot (resolution estimation)

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