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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
KeywordsComplex Kinase / SIGNALING PROTEIN
Function / homology
Function and homology information


CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / Signalling to p38 via RIT and RIN / myeloid progenitor cell differentiation / head morphogenesis / ARMS-mediated activation / endothelial cell apoptotic process / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling ...CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / Signalling to p38 via RIT and RIN / myeloid progenitor cell differentiation / head morphogenesis / ARMS-mediated activation / endothelial cell apoptotic process / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / negative regulation of fibroblast migration / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / positive regulation of axonogenesis / regulation of T cell differentiation / mitogen-activated protein kinase kinase binding / Negative feedback regulation of MAPK pathway / Frs2-mediated activation / positive regulation of axon regeneration / stress fiber assembly / face development / MAP kinase kinase activity / synaptic vesicle exocytosis / thyroid gland development / somatic stem cell population maintenance / MAP kinase kinase kinase activity / postsynaptic modulation of chemical synaptic transmission / negative regulation of endothelial cell apoptotic process / response to cAMP / positive regulation of peptidyl-serine phosphorylation / positive regulation of stress fiber assembly / positive regulation of substrate adhesion-dependent cell spreading / ERK1 and ERK2 cascade / substrate adhesion-dependent cell spreading / cellular response to calcium ion / thymus development / animal organ morphogenesis / RAF activation / cellular response to nerve growth factor stimulus / Spry regulation of FGF signaling / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / visual learning / response to peptide hormone / centriolar satellite / long-term synaptic potentiation / epidermal growth factor receptor signaling pathway / Signaling by RAF1 mutants / Negative regulation of MAPK pathway / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / Signaling by BRAF and RAF1 fusions / MAPK cascade / cellular response to xenobiotic stimulus / presynapse / T cell receptor signaling pathway / regulation of cell population proliferation / T cell differentiation in thymus / cell body / scaffold protein binding / negative regulation of neuron apoptotic process / eukaryotic translation initiation factor 2alpha kinase activity / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein kinase activity / histone H2AS1 kinase activity / histone H3T6 kinase activity / histone H3T11 kinase activity / histone H3T45 kinase activity / positive regulation of ERK1 and ERK2 cascade / non-specific serine/threonine protein kinase / postsynapse / protein kinase activity / neuron projection / ciliary basal body / cilium / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / intracellular membrane-bounded organelle / calcium ion binding / positive regulation of gene expression / protein-containing complex binding / negative regulation of apoptotic process / glutamatergic synapse / mitochondrion / zinc ion binding
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) ...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 / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / 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 protein zeta isoform X1 / Serine/threonine-protein kinase B-raf
Similarity search - Component
Biological speciesHomo sapiens (human) / Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKondo Y / Ognjenovic 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-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing 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
  • Download
  • 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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 212. Å		1.06 Å/pix.
x 200 pix.
= 212. Å		1.06 Å/pix.
x 200 pix.
= 212. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.056520123 - 0.10374748
Average (Standard dev.)-0.0000021298338 (±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)

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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: Spodoptera aff. frugiperda 1 BOLD-2017 (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

UniProtKB: 14-3-3 protein zeta isoform X1

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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

UniProtKB: Serine/threonine-protein kinase B-raf

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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-HCl
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 / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 278 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
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
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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