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- EMDB-23813: Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD -

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Basic information

Entry
Database: EMDB / ID: EMD-23813
TitleAutoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD
Map data
Sample
  • Complex: Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD
    • Complex: B-raf
      • Protein or peptide: Serine/threonine-protein kinase B-raf
    • Complex: MEK
      • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
    • Complex: 14-3-3 protein zeta/delta
      • Protein or peptide: 14-3-3 protein zeta/delta
  • Ligand: ZINC ION
  • Ligand: N-(3-fluoro-4-{[4-methyl-2-oxo-7-(pyrimidin-2-yloxy)-2H-chromen-3-yl]methyl}pyridin-2-yl)-N'-methylsulfuric diamide
KeywordsB-Raf / MEK / 14-3-3 / B-Raf complex / B-Raf monomer / Inactive B-Raf / Serine/threonine-protein kinase B-raf / RBD / signaling protein / SIGNALING PROTEIN-TRANSFERASE complex
Function / homology
Function and homology information


epithelial cell proliferation involved in lung morphogenesis / synaptic target recognition / positive regulation of endodermal cell differentiation / Golgi reassembly / JUN kinase kinase activity / CD4-positive, alpha-beta T cell differentiation / regulation of axon regeneration / NOTCH4 Activation and Transmission of Signal to the Nucleus / mitogen-activated protein kinase kinase / establishment of Golgi localization ...epithelial cell proliferation involved in lung morphogenesis / synaptic target recognition / positive regulation of endodermal cell differentiation / Golgi reassembly / JUN kinase kinase activity / CD4-positive, alpha-beta T cell differentiation / regulation of axon regeneration / NOTCH4 Activation and Transmission of Signal to the Nucleus / mitogen-activated protein kinase kinase / establishment of Golgi localization / placenta blood vessel development / MAP-kinase scaffold activity / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / labyrinthine layer development / negative regulation of synaptic vesicle exocytosis / cerebellar cortex formation / type B pancreatic cell proliferation / Signalling to p38 via RIT and RIN / respiratory system process / myeloid progenitor cell differentiation / head morphogenesis / regulation of synapse maturation / ARMS-mediated activation / tube formation / endothelial cell apoptotic process / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / negative regulation of fibroblast migration / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / positive regulation of axonogenesis / negative regulation of protein localization to nucleus / regulation of Golgi inheritance / regulation of T cell differentiation / trachea formation / mitogen-activated protein kinase kinase binding / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / KSRP (KHSRP) binds and destabilizes mRNA / regulation of stress-activated MAPK cascade / MAPK3 (ERK1) activation / GP1b-IX-V activation signalling / Frs2-mediated activation / ERBB2-ERBB3 signaling pathway / positive regulation of axon regeneration / stress fiber assembly / endodermal cell differentiation / face development / MAP kinase kinase activity / Bergmann glial cell differentiation / synaptic vesicle exocytosis / Uptake and function of anthrax toxins / thyroid gland development / Regulation of localization of FOXO transcription factors / somatic stem cell population maintenance / protein kinase activator activity / Interleukin-3, Interleukin-5 and GM-CSF signaling / phosphoserine residue binding / MAP kinase kinase kinase activity / Activation of BAD and translocation to mitochondria / positive regulation of protein serine/threonine kinase activity / postsynaptic modulation of chemical synaptic transmission / negative regulation of endothelial cell apoptotic process / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / protein targeting / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / Schwann cell development / regulation of ERK1 and ERK2 cascade / cellular response to glucose starvation / keratinocyte differentiation / response to cAMP / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / positive regulation of peptidyl-serine phosphorylation / positive regulation of stress fiber assembly / negative regulation of TORC1 signaling / positive regulation of substrate adhesion-dependent cell spreading / myelination / Transcriptional and post-translational regulation of MITF-M expression and activity / ERK1 and ERK2 cascade / protein serine/threonine/tyrosine kinase activity / substrate adhesion-dependent cell spreading / protein sequestering activity / insulin-like growth factor receptor signaling pathway / cellular response to calcium ion / negative regulation of innate immune response / hippocampal mossy fiber to CA3 synapse / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / thymus development / animal organ morphogenesis / Signal transduction by L1 / Translocation of SLC2A4 (GLUT4) to the plasma membrane / TP53 Regulates Metabolic Genes / cell motility / Deactivation of the beta-catenin transactivating complex / lung development / RAF activation
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. ...: / 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. / Protein kinase domain / 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
Serine/threonine-protein kinase B-raf / 14-3-3 protein zeta/delta / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsMartinez Fiesco JA / Ping Z
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 011744 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 010329 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding.
Authors: Juliana A Martinez Fiesco / David E Durrant / Deborah K Morrison / Ping Zhang /
Abstract: RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present ...RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-3:MEK and BRAF:14-3-3 complexes, and an inhibitor-bound, dimeric BRAF:14-3-3 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.
History
DepositionApr 9, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0208
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0208
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mfd
  • Surface level: 0.0208
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mfd
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23813.png

Downloads & links

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Map

FileDownload / File: emd_23813.map.gz / Format: CCP4 / Size: 22.2 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 180 pix.
= 190.44 Å		1.06 Å/pix.
x 180 pix.
= 190.44 Å		1.06 Å/pix.
x 180 pix.
= 190.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.058 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0208 / Movie #1: 0.0208
Minimum - Maximum-0.106111236 - 0.18190569
Average (Standard dev.)0.00043417784 (±0.004040884)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 190.43999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0581.0581.058
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z190.440190.440190.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.1060.1820.000

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Supplemental data

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Sample components

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Entire : Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD

EntireName: Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD
Components
  • Complex: Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD
    • Complex: B-raf
      • Protein or peptide: Serine/threonine-protein kinase B-raf
    • Complex: MEK
      • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
    • Complex: 14-3-3 protein zeta/delta
      • Protein or peptide: 14-3-3 protein zeta/delta
  • Ligand: ZINC ION
  • Ligand: N-(3-fluoro-4-{[4-methyl-2-oxo-7-(pyrimidin-2-yloxy)-2H-chromen-3-yl]methyl}pyridin-2-yl)-N'-methylsulfuric diamide

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Supramolecule #1: Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD

SupramoleculeName: Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBD
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: B-raf

SupramoleculeName: B-raf / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: MEK

SupramoleculeName: MEK / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: 14-3-3 protein zeta/delta

SupramoleculeName: 14-3-3 protein zeta/delta / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Serine/threonine-protein kinase B-raf

MacromoleculeName: Serine/threonine-protein kinase B-raf / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 84.697695 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAALSGGGGG GAEPGQALFN GDMEPEAGAG AGAAASSAAD PAIPEEVWNI KQMIKLTQEH IEALLDKFGG EHNPPSIYLE AYEEYTSKL DALQQREQQL LESLGNGTDF SVSSSASMDT VTSSSSSSLS VLPSSLSVFQ NPTDVARSNP KSPQKPIVRV F LPNKQRTV ...String:
MAALSGGGGG GAEPGQALFN GDMEPEAGAG AGAAASSAAD PAIPEEVWNI KQMIKLTQEH IEALLDKFGG EHNPPSIYLE AYEEYTSKL DALQQREQQL LESLGNGTDF SVSSSASMDT VTSSSSSSLS VLPSSLSVFQ NPTDVARSNP KSPQKPIVRV F LPNKQRTV VPARCGVTVR DSLKKALMMR GLIPECCAVY RIQDGEKKPI GWDTDISWLT GEELHVEVLE NVPLTTHNFV RK TFFTLAF CDFCRKLLFQ GFRCQTCGYK FHQRCSTEVP LMCVNYDQLD LLFVSKFFEH HPIPQEEASL AETALTSGSS PSA PASDSI GPQILTSPSP SKSIPIPQPF RPADEDHRNQ FGQRDRSS(SEP)A PNVHINTIEP VNIDDLIRDQ GFRGDGGSTT GLSATPPAS LPGSLTNVKA LQKSPGPQRE RKSSSSSEDR NRMKTLGRRD SSDDWEIPDG QITVGQRIGS GSFGTVYKGK W HGDVAVKM LNVTAPTPQQ LQAFKNEVGV LRKTRHVNIL LFMGYSTKPQ LAIVTQWCEG SSLYHHLHII ETKFEMIKLI DI ARQTAQG MDYLHAKSII HRDLKSNNIF LHEDLTVKIG DFGLATVKSR WSGSHQFEQL SGSILWMAPE VIRMQDKNPY SFQ SDVYAF GIVLYELMTG QLPYSNINNR DQIIFMVGRG YLSPDLSKVR SNCPKAMKRL MAECLKKKRD ERPLFPQILA SIEL LARSL PKIHRSA(SEP)EP SLNRAGFQTE DFSLYACASP KTPIQAGGYG AFPVH

UniProtKB: Serine/threonine-protein kinase B-raf

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Macromolecule #2: Dual specificity mitogen-activated protein kinase kinase 1

MacromoleculeName: Dual specificity mitogen-activated protein kinase kinase 1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: mitogen-activated protein kinase kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.493938 KDa
SequenceString: MPKKKPTPIQ LNPAPDGSAV NGTSSAETNL EALQKKLEEL ELDEQQRKRL EAFLTQKQKV GELKDDDFEK ISELGAGNGG VVFKVSHKP SGLVMARKLI HLEIKPAIRN QIIRELQVLH ECNSPYIVGF YGAFYSDGEI SICMEHMDGG SLDQVLKKAG R IPEQILGK ...String:
MPKKKPTPIQ LNPAPDGSAV NGTSSAETNL EALQKKLEEL ELDEQQRKRL EAFLTQKQKV GELKDDDFEK ISELGAGNGG VVFKVSHKP SGLVMARKLI HLEIKPAIRN QIIRELQVLH ECNSPYIVGF YGAFYSDGEI SICMEHMDGG SLDQVLKKAG R IPEQILGK VSIAVIKGLT YLREKHKIMH RDVKPSNILV NSRGEIKLCD FGVSGQLIDS MANSFVGTRS YMSPERLQGT HY SVQSDIW SMGLSLVEMA VGRYPIPPPD AKELELMFGC QVEGDAAETP PRPRTPGRPL SSYGMDSRPP MAIFELLDYI VNE PPPKLP SGVFSLEFQD FVNKCLIKNP AERADLKQLM VHAFIKRSDA EEVDFAGWLC STIGLNQPST PTHAAGV

UniProtKB: Dual specificity mitogen-activated protein kinase kinase 1

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Macromolecule #3: 14-3-3 protein zeta/delta

MacromoleculeName: 14-3-3 protein zeta/delta / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.777092 KDa
SequenceString: MDKNELVQKA KLAEQAERYD DMAACMKSVT EQGAELSNEE RNLLSVAYKN VVGARRSSWR VVSSIEQKTE GAEKKQQMAR EYREKIETE LRDICNDVLS LLEKFLIPNA SQAESKVFYL KMKGDYYRYL AEVAAGDDKK GIVDQSQQAY QEAFEISKKE M QPTHPIRL ...String:
MDKNELVQKA KLAEQAERYD DMAACMKSVT EQGAELSNEE RNLLSVAYKN VVGARRSSWR VVSSIEQKTE GAEKKQQMAR EYREKIETE LRDICNDVLS LLEKFLIPNA SQAESKVFYL KMKGDYYRYL AEVAAGDDKK GIVDQSQQAY QEAFEISKKE M QPTHPIRL GLALNFSVFY YEILNSPEKA CSLAKTAFDE AIAELDTLSE ESYKDSTLIM QLLRDNLTLW TSDTQGDEAE AG EGGEN

UniProtKB: 14-3-3 protein zeta/delta

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: N-(3-fluoro-4-{[4-methyl-2-oxo-7-(pyrimidin-2-yloxy)-2H-chromen-3...

MacromoleculeName: N-(3-fluoro-4-{[4-methyl-2-oxo-7-(pyrimidin-2-yloxy)-2H-chromen-3-yl]methyl}pyridin-2-yl)-N'-methylsulfuric diamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: CHU
Molecular weightTheoretical: 471.462 Da
Chemical component information

ChemComp-CHU:
N-(3-fluoro-4-{[4-methyl-2-oxo-7-(pyrimidin-2-yloxy)-2H-chromen-3-yl]methyl}pyridin-2-yl)-N'-methylsulfuric diamide

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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:
ConcentrationNameFormula
20.0 mMTris
200.0 mMSodium chlorideNaCl
10.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: 20mAmp
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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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: 57.0 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: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 142852
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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