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- EMDB-16660: cryo-EM Structure of Craf:14-3-3:Mek1 -

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Entry
Database: EMDB / ID: EMD-16660
Titlecryo-EM Structure of Craf:14-3-3:Mek1
Map data
Sample
  • Complex: Complex of dimer phosphorylated C-raf kinase domain with 14-3-3 dimer and 2 subunits of Mek1 containing ligand GDC-0623
    • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Protein or peptide: 14-3-3 protein zeta isoform X1
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
  • Ligand: 2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridin-4-yl)-1H-pyrazol-1-yl}ethanol
KeywordsKinase / STRUCTURAL PROTEIN
Function / homology
Function and homology information


death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / negative regulation of homotypic cell-cell adhesion / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / regulation of vascular associated smooth muscle contraction / intermediate filament cytoskeleton organization / mitogen-activated protein kinase kinase / Golgi inheritance / placenta blood vessel development ...death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / negative regulation of homotypic cell-cell adhesion / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / regulation of vascular associated smooth muscle contraction / intermediate filament cytoskeleton organization / mitogen-activated protein kinase kinase / Golgi inheritance / placenta blood vessel development / MAP-kinase scaffold activity / positive regulation of muscle contraction / regulation of axon regeneration / cerebellar cortex formation / labyrinthine layer development / regulation of Rho protein signal transduction / melanosome transport / type B pancreatic cell proliferation / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / vesicle transport along microtubule / positive regulation of axonogenesis / positive regulation of Ras protein signal transduction / insulin secretion involved in cellular response to glucose stimulus / regulation of Golgi inheritance / mitogen-activated protein kinase kinase kinase binding / central nervous system neuron differentiation / triglyceride homeostasis / trachea formation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / IFNG signaling activates MAPKs / regulation of stress-activated MAPK cascade / Frs2-mediated activation / GP1b-IX-V activation signalling / MAPK3 (ERK1) activation / ERBB2-ERBB3 signaling pathway / neurotrophin TRK receptor signaling pathway / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / pseudopodium / face development / endodermal cell differentiation / MAP kinase kinase activity / Bergmann glial cell differentiation / positive regulation of ATP biosynthetic process / regulation of cell differentiation / thyroid gland development / Uptake and function of anthrax toxins / positive regulation of protein serine/threonine kinase activity / extrinsic apoptotic signaling pathway via death domain receptors / somatic stem cell population maintenance / protein kinase activator activity / positive regulation of peptidyl-serine phosphorylation / MAP kinase kinase kinase activity / type II interferon-mediated signaling pathway / negative regulation of protein-containing complex assembly / Schwann cell development / response to axon injury / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / keratinocyte differentiation / neuron projection morphogenesis / response to muscle stretch / ERK1 and ERK2 cascade / myelination / positive regulation of autophagy / protein serine/threonine/tyrosine kinase activity / CD209 (DC-SIGN) signaling / dendrite cytoplasm / insulin-like growth factor receptor signaling pathway / response to glucocorticoid / MAP3K8 (TPL2)-dependent MAPK1/3 activation / thymus development / adenylate cyclase activator activity / protein serine/threonine kinase activator activity / Signal transduction by L1 / cell motility / positive regulation of transcription elongation by RNA polymerase II / wound healing / RAF activation / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / small GTPase binding / Stimuli-sensing channels / neuron differentiation / chemotaxis / 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 / cellular senescence / Signaling by BRAF and RAF1 fusions / insulin receptor signaling pathway / late endosome / MAPK cascade / heart development / response to oxidative stress / protein tyrosine kinase activity
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 / 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
14-3-3 protein zeta / RAF proto-oncogene serine/threonine-protein kinase / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Spodoptera frugiperda (fall armyworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsDedden D / Ulrich G
Funding support Germany, 1 items
OrganizationGrant numberCountry
Other private Germany
CitationJournal: J Mol Biol / Year: 2024
Title: Cryo-EM Structures of CRAF/14-3-3 and CRAF/14-3-3/MEK1 Complexes.
Authors: Dirk Dedden / Julius Nitsche / Elisabeth V Schneider / Maren Thomsen / Daniel Schwarz / Birgitta Leuthner / Ulrich Grädler /
Abstract: RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) ...RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF/14-3-3 at 3.4 Å resolution and CRAF/14-3-3/MEK1 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF/14-3-3 and CRAF/14-3-3/MEK1 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF/14-3-3/MEK1 complex.
History
DepositionFeb 7, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16660.png

Downloads & links

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Map

FileDownload / File: emd_16660.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 256 pix.
= 233.898 Å		0.91 Å/pix.
x 256 pix.
= 233.898 Å		0.91 Å/pix.
x 256 pix.
= 233.898 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91366 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-0.01917762 - 2.184309
Average (Standard dev.)0.0025782855 (±0.036138844)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 233.89798 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16660_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_16660_half_map_2.map
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Sample components

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Entire : Complex of dimer phosphorylated C-raf kinase domain with 14-3-3 d...

EntireName: Complex of dimer phosphorylated C-raf kinase domain with 14-3-3 dimer and 2 subunits of Mek1 containing ligand GDC-0623
Components
  • Complex: Complex of dimer phosphorylated C-raf kinase domain with 14-3-3 dimer and 2 subunits of Mek1 containing ligand GDC-0623
    • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Protein or peptide: 14-3-3 protein zeta isoform X1
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
  • Ligand: 2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridin-4-yl)-1H-pyrazol-1-yl}ethanol

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Supramolecule #1: Complex of dimer phosphorylated C-raf kinase domain with 14-3-3 d...

SupramoleculeName: Complex of dimer phosphorylated C-raf kinase domain with 14-3-3 dimer and 2 subunits of Mek1 containing ligand GDC-0623
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: RAF proto-oncogene serine/threonine-protein kinase

MacromoleculeName: RAF proto-oncogene serine/threonine-protein kinase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73.184477 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEHIQGAWKT ISNGFGFKDA VFDGSSCISP TIVQQFGYQR RASDDGKLTD PSKTSNTIRV FLPNKQRTVV NVRNGMSLHD CLMKALKVR GLQPECCAVF RLLHEHKGKK ARLDWNTDAA SLIGEELQVD FLDHVPLTTH NFARKTFLKL AFCDICQKFL L NGFRCQTC ...String:
MEHIQGAWKT ISNGFGFKDA VFDGSSCISP TIVQQFGYQR RASDDGKLTD PSKTSNTIRV FLPNKQRTVV NVRNGMSLHD CLMKALKVR GLQPECCAVF RLLHEHKGKK ARLDWNTDAA SLIGEELQVD FLDHVPLTTH NFARKTFLKL AFCDICQKFL L NGFRCQTC GYKFHEHCST KVPTMCVDWS NIRQLLLFPN STIGDSGVPA LPSLTMRRMR ESVSRMPVSS QHRYSTPHAF TF NTSSPSS EGSLSQRQRS TSTPNVHMVS TTLPVDSRMI EDAIRSHSES ASPSALSSSP NNLSPTGWSQ PKTPVPAQRE RAP VSGTQE KNKIRPRGQR DSSYDWEIEA SEVMLSTRIG SGSFGTVYKG KWHGDVAVKI LKVVDPTPEQ FQAFRNEVAV LRKT RHVNI LLFMGYMTKD NLAIVTQWCE GSSLYKHLHV QETKFQMFQL IDIARQTAQG MDYLHAKNII HRDMKSNNIF LHEGL TVKI GDFGLATVKS RWSGSQQVEQ PTGSVLWMAP EVIRMQDNNP FSFQSDVYSY GIVLYELMTG ELPYSHINNR DQIIFM VGR GYASPDLSKL YKNCPKAMKR LVADCVKKVK EERPLFPQIL SSIELLQHSL PKINRSA(SEP)EP SLHRAAHTED INA CTLTTS PRLPVF

UniProtKB: RAF proto-oncogene serine/threonine-protein kinase

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Macromolecule #2: 14-3-3 protein zeta isoform X1

MacromoleculeName: 14-3-3 protein zeta isoform X1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)
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

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Macromolecule #3: Dual specificity mitogen-activated protein kinase kinase 1

MacromoleculeName: Dual specificity mitogen-activated protein kinase kinase 1
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: mitogen-activated protein kinase kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.461938 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
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 FGVSGQLIDA MANAFVGTRS 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 #4: 2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridin...

MacromoleculeName: 2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridin-4-yl)-1H-pyrazol-1-yl}ethanol
type: ligand / ID: 4 / Number of copies: 2 / Formula: 29L
Molecular weightTheoretical: 334.372 Da
Chemical component information

ChemComp-29L:
2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridin-4-yl)-1H-pyrazol-1-yl}ethanol

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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

Concentration0.4 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 2732 / Average exposure time: 7.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: NONE
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 79383
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8chf:
cryo-EM Structure of Craf:14-3-3:Mek1

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