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- EMDB-23813: Autoinhibited BRAF:(14-3-3)2:MEK complex with the BRAF RBD resolved -

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Basic information

Entry
Database: EMDB / ID: EMD-23813
TitleAutoinhibited BRAF:(14-3-3)2:MEK complex with the BRAF RBD resolved
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
Function / homology
Function and homology information


Golgi reassembly / synaptic target recognition / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / respiratory system process / NOTCH4 Activation and Transmission of Signal to the Nucleus / regulation of synapse maturation / CD4-positive, alpha-beta T cell differentiation / placenta blood vessel development / trehalose metabolism in response to stress ...Golgi reassembly / synaptic target recognition / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / respiratory system process / NOTCH4 Activation and Transmission of Signal to the Nucleus / regulation of synapse maturation / CD4-positive, alpha-beta T cell differentiation / placenta blood vessel development / trehalose metabolism in response to stress / regulation of axon regeneration / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / mitogen-activated protein kinase kinase / establishment of Golgi localization / negative regulation of synaptic vesicle exocytosis / labyrinthine layer development / type B pancreatic cell proliferation / MAP-kinase scaffold activity / Signalling to p38 via RIT and RIN / cerebellar cortex formation / head morphogenesis / myeloid progenitor cell differentiation / tube formation / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / endothelial cell apoptotic process / Signaling by MAP2K mutants / Rap1 signalling / negative regulation of fibroblast migration / positive regulation of glucose transmembrane transport / establishment of protein localization to membrane / negative regulation of protein localization to nucleus / regulation of Golgi inheritance / mitogen-activated protein kinase kinase binding / trachea formation / regulation of T cell differentiation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / KSRP (KHSRP) binds and destabilizes mRNA / positive regulation of axonogenesis / regulation of stress-activated MAPK cascade / Frs2-mediated activation / GP1b-IX-V activation signalling / ERBB2-ERBB3 signaling pathway / stress fiber assembly / positive regulation of axon regeneration / protein kinase activator activity / endodermal cell differentiation / face development / MAPK3 (ERK1) activation / synaptic vesicle exocytosis / somatic stem cell population maintenance / Bergmann glial cell differentiation / MAP kinase kinase activity / thyroid gland development / Uptake and function of anthrax toxins / Regulation of localization of FOXO transcription factors / Interleukin-3, Interleukin-5 and GM-CSF signaling / phosphoserine residue binding / MAP kinase kinase kinase activity / Activation of BAD and translocation to mitochondria / protein targeting / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / cellular response to glucose starvation / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / Schwann cell development / negative regulation of endothelial cell apoptotic process / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / positive regulation of substrate adhesion-dependent cell spreading / RHO GTPases activate PKNs / keratinocyte differentiation / positive regulation of stress fiber assembly / negative regulation of TORC1 signaling / response to cAMP / protein sequestering activity / cellular response to calcium ion / ERK1 and ERK2 cascade / negative regulation of innate immune response / myelination / protein serine/threonine/tyrosine kinase activity / hippocampal mossy fiber to CA3 synapse / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / substrate adhesion-dependent cell spreading / regulation of ERK1 and ERK2 cascade / insulin-like growth factor receptor signaling pathway / cellular response to nerve growth factor stimulus / thymus development / Signal transduction by L1 / Deactivation of the beta-catenin transactivating complex / Translocation of SLC2A4 (GLUT4) to the plasma membrane / cell motility / long-term synaptic potentiation / TP53 Regulates Metabolic Genes / Negative regulation of NOTCH4 signaling / animal organ morphogenesis / Spry regulation of FGF signaling / RAF activation / lung development
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. / 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) / Human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsMartinez Fiesco JA / Ping Z / Durrant DE / Morrison DK
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-
UpdateFeb 9, 2022-
Current statusFeb 9, 2022Processing 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)
Voxel sizeX=Y=Z: 1.058 Å
Density
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)
Recombinant expressionOrganism: 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

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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: Human (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

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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: Human (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

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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 / 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

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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