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- EMDB-48402: Cryo-EM structure of CRAF/MEK1 complex (kinase domain, CRAF Y340D... -

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Entry
Database: EMDB / ID: EMD-48402
TitleCryo-EM structure of CRAF/MEK1 complex (kinase domain, CRAF Y340D/Y341D mutant)
Map data
Sample
  • Complex: CRAF/MEK1/14-3-3 complex
    • Complex: CRAF
      • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Complex: MEK1
      • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide
KeywordsCRAF-MEK1-14-3-3 complex / MAPK pathway / TRANSFERASE
Function / homology
Function and homology information


negative regulation of homotypic cell-cell adhesion / regulation of vascular associated smooth muscle contraction / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / mitogen-activated protein kinase kinase / melanosome transport / Golgi inheritance / MAP kinase scaffold activity / positive regulation of muscle contraction / regulation of Rho protein signal transduction / Signaling by MAP2K mutants ...negative regulation of homotypic cell-cell adhesion / regulation of vascular associated smooth muscle contraction / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / mitogen-activated protein kinase kinase / melanosome transport / Golgi inheritance / MAP kinase scaffold activity / positive regulation of muscle contraction / regulation of Rho protein signal transduction / 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 / regulation of Golgi inheritance / mitogen-activated protein kinase kinase kinase binding / positive regulation of protein serine/threonine kinase activity / regulation of early endosome to late endosome transport / triglyceride homeostasis / regulation of stress-activated MAPK cascade / Negative feedback regulation of MAPK pathway / IFNG signaling activates MAPKs / GP1b-IX-V activation signalling / Frs2-mediated activation / MAPK3 (ERK1) activation / ERBB2-ERBB3 signaling pathway / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / MAP kinase kinase activity / regulation of cell differentiation / pseudopodium / response to axon injury / positive regulation of ATP biosynthetic process / ERK1 and ERK2 cascade / neuromuscular junction development / Uptake and function of anthrax toxins / positive regulation of peptidyl-serine phosphorylation / MAP kinase kinase kinase activity / type II interferon-mediated signaling pathway / protein kinase activator activity / Schwann cell development / serine/threonine protein kinase complex / negative regulation of protein-containing complex assembly / myelination / insulin-like growth factor receptor signaling pathway / protein serine/threonine/tyrosine kinase activity / positive regulation of autophagy / neuron projection morphogenesis / CD209 (DC-SIGN) signaling / response to glucocorticoid / protein serine/threonine kinase activator activity / dendrite cytoplasm / adenylate cyclase activator activity / wound healing / Signal transduction by L1 / MAP3K8 (TPL2)-dependent MAPK1/3 activation / positive regulation of transcription elongation by RNA polymerase II / RAF activation / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / chemotaxis / cellular senescence / insulin receptor signaling pathway / small GTPase binding / Stimuli-sensing channels / 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 / Negative regulation of MAPK pathway / Signaling by BRAF and RAF1 fusions / neuron differentiation / late endosome / microtubule / ciliary basal body / response to oxidative stress / protein tyrosine kinase activity / scaffold protein binding / cell cortex / early endosome / regulation of apoptotic process / perikaryon / positive regulation of ERK1 and ERK2 cascade / protein kinase activity / protein phosphorylation / positive regulation of MAPK cascade / non-specific serine/threonine protein kinase / mitochondrial outer membrane / postsynaptic density / apoptotic process / positive regulation of cell migration / negative regulation of cell population proliferation / negative regulation of gene expression / protein serine kinase activity / axon / focal adhesion / protein serine/threonine kinase activity / centrosome / positive regulation of gene expression / negative regulation of apoptotic process
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 / 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
RAF proto-oncogene serine/threonine-protein kinase / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsJang DM / Jeon H / Eck MJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R35CA242461-06 United States
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM structures of CRAF/MEK1/14-3-3 complexes in autoinhibited and open-monomer states reveal features of RAF regulation.
Authors: Dong Man Jang / Kayla Boxer / Byung Hak Ha / Emre Tkacik / Talya Levitz / Shaun Rawson / Rebecca J Metivier / Anna Schmoker / Hyesung Jeon / Michael J Eck /
Abstract: CRAF (RAF1) is one of three RAF-family kinases that initiate MAP kinase signaling in response to activated RAS and is essential for oncogenic signaling from mutant KRAS. Like BRAF, CRAF is regulated ...CRAF (RAF1) is one of three RAF-family kinases that initiate MAP kinase signaling in response to activated RAS and is essential for oncogenic signaling from mutant KRAS. Like BRAF, CRAF is regulated by 14-3-3 engagement and by intramolecular autoinhibitory interactions of its N-terminal regulatory region. Unlike BRAF, it is thought to require tyrosine phosphorylation in its N-terminal acidic (NtA) motif for full catalytic activation. Here we describe cryo-EM reconstructions of full-length CRAF in complex with MEK1 and a 14-3-3 dimer. These structures reveal a fully autoinhibited conformation analogous to that observed for BRAF and two "open monomer" states in which the inhibitory interactions of the CRD and 14-3-3 dimer are released or rearranged, but the kinase domain remains inactive. Structure-function studies of the NtA motif indicate that phosphorylation or acidic mutations in this segment increase catalytic activity by destabilizing the inactive conformation of the kinase domain. Collectively, these studies provide a structural foundation for understanding the shared and unique regulatory features of CRAF and will inform efforts to selectively block CRAF signaling in cancer.
History
DepositionDec 20, 2024-
Header (metadata) releaseSep 10, 2025-
Map releaseSep 10, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48402.png

Downloads & links

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Map

FileDownload / File: emd_48402.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 288 pix.
= 210.24 Å		0.73 Å/pix.
x 288 pix.
= 210.24 Å		0.73 Å/pix.
x 288 pix.
= 210.24 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.73 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.06537376 - 0.13357715
Average (Standard dev.)-0.000026412987 (±0.0028951697)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 210.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48402_msk_1.map
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Half map: #2

Fileemd_48402_half_map_1.map
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Half map: #1

Fileemd_48402_half_map_2.map
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Sample components

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Entire : CRAF/MEK1/14-3-3 complex

EntireName: CRAF/MEK1/14-3-3 complex
Components
  • Complex: CRAF/MEK1/14-3-3 complex
    • Complex: CRAF
      • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Complex: MEK1
      • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide

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Supramolecule #1: CRAF/MEK1/14-3-3 complex

SupramoleculeName: CRAF/MEK1/14-3-3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55 KDa

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Supramolecule #2: CRAF

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

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Supramolecule #3: MEK1

SupramoleculeName: MEK1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
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: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 76.881609 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSSHHHHHH SAVDENLYFQ GGMEHIQGAW KTISNGFGFK DAVFDGSSCI SPTIVQQFGY QRRASDDGKL TDPSKTSNTI RVFLPNKQR TVVNVRNGMS LHDCLMKALK VRGLQPECCA VFRLLHEHKG KKARLDWNTD AASLIGEELQ VDFLDHVPLT T HNFARKTF ...String:
MGSSHHHHHH SAVDENLYFQ GGMEHIQGAW KTISNGFGFK DAVFDGSSCI SPTIVQQFGY QRRASDDGKL TDPSKTSNTI RVFLPNKQR TVVNVRNGMS LHDCLMKALK VRGLQPECCA VFRLLHEHKG KKARLDWNTD AASLIGEELQ VDFLDHVPLT T HNFARKTF LKLAFCDICR KFLLNGFRCQ TCGYKFHEHC STKVPTMCVD WSNIRQLLLF PNSTIGDSGV PALPSLTMRR MR ESVSRMP VSSQHRYSTP HAFTFNTSSP SSEGSLSQRQ RSTSTPNVHM VSTTLPVDSR MIEDAIRSHS ESASPSALSS SPN NLSPTG WSQPKTPVPA QRERAPVSGT QEKNKIRPRG QRDSSDDWEI EASEVMLSTR IGSGSFGTVY KGKWHGDVAV KILK VVDPT PEQFQAFRNE VAVLRKTRHV NILLFMGYMT KDNLAIVTQW CEGSSLYKHL HVQETKFQMF QLIDIARQTA QGMDY LHAK NIIHRDMKSN NIFLHEGLTV KIGDFGLATV KSRWSGSQQV EQPTGSVLWM APEVIRMQDN NPFSFQSDVY SYGIVL YEL MTGELPYSHI NNRDQIIFMV GRGYASPDLS KLYKNCPKAM KRLVAECVKK VKEERPLFPQ ILSSIELLQH SLPKINR SA SEPSLHRAAH TEDINACTLT TSPRLPVFVP AWSHPQFEK

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

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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: 45.934543 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSSHHHHHH SAVDENLYFQ GGMPKKKPTP IQLNPAPDGS AVNGTSSAET NLEALQKKLE ELELDEQQRK RLEAFLTQKQ KVGELKDDD FEKISELGAG NGGVVFKVSH KPSGLVMARK LIHLEIKPAI RNQIIRELQV LHECNSPYIV GFYGAFYSDG E ISICMEHM ...String:
MGSSHHHHHH SAVDENLYFQ GGMPKKKPTP IQLNPAPDGS AVNGTSSAET NLEALQKKLE ELELDEQQRK RLEAFLTQKQ KVGELKDDD FEKISELGAG NGGVVFKVSH KPSGLVMARK LIHLEIKPAI RNQIIRELQV LHECNSPYIV GFYGAFYSDG E ISICMEHM DGGSLDQVLK KAGRIPEQIL GKVSIAVIKG LTYLREKHKI MHRDVKPSNI LVNSRGEIKL CDFGVSGQLI DA MANAFVG TRSYMSPERL QGTHYSVQSD IWSMGLSLVE MAVGRYPIPP PDAKELELMF GCQVEGDAAE TPPRPRTPGR PLS SYGMDS RPPMAIFELL DYIVNEPPPK LPSGVFSLEF QDFVNKCLIK NPAERADLKQ LMVHAFIKRS DAEEVDFAGW LCST IGLNQ PSTPTHAAGV

UniProtKB: Dual specificity mitogen-activated protein kinase kinase 1

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 2 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a...

MacromoleculeName: 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: LCJ
Molecular weightTheoretical: 456.21 Da
Chemical component information

ChemComp-LCJ:
5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide

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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
Details: 50 mM Tris pH 7.5, 150 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP, 2 uM ATPgS, 1 uM GDC0623
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON I (4k x 4k) / Number grids imaged: 1 / Number real images: 7289 / Average electron dose: 51.26 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
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
PDB model - PDB ID:

9ay7

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Details: The final resolution was calculated without a mask / Number images used: 175214
Initial angle assignmentType: OTHER / Details: Ab-initio reconstruction in CryoSPARC
Final angle assignmentType: OTHER / Details: NU-refinement in CryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-9mms:
Cryo-EM structure of CRAF/MEK1 complex (kinase domain, CRAF Y340D/Y341D mutant)

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