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

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Basic information

Entry
Database: PDB / ID: 9mms
TitleCryo-EM structure of CRAF/MEK1 complex (kinase domain, CRAF Y340D/Y341D mutant)
Components
  • Dual specificity mitogen-activated protein kinase kinase 1
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsTRANSFERASE / CRAF-MEK1-14-3-3 complex / MAPK pathway
Function / homology
Function and homology information


insulin secretion involved in cellular response to glucose stimulus / death-inducing signaling complex assembly / 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 / MAP kinase scaffold activity / type B pancreatic cell proliferation ...insulin secretion involved in cellular response to glucose stimulus / death-inducing signaling complex assembly / 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 / MAP kinase scaffold activity / type B pancreatic cell proliferation / positive regulation of muscle contraction / melanosome transport / 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 / triglyceride homeostasis / regulation of early endosome to late endosome transport / Negative feedback regulation of MAPK pathway / neurotrophin TRK receptor signaling pathway / regulation of stress-activated MAPK cascade / IFNG signaling activates MAPKs / GP1b-IX-V activation signalling / Frs2-mediated activation / face development / MAPK3 (ERK1) activation / thyroid gland development / ERBB2-ERBB3 signaling pathway / MAP kinase kinase activity / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / positive regulation of protein serine/threonine kinase activity / extrinsic apoptotic signaling pathway via death domain receptors / pseudopodium / somatic stem cell population maintenance / regulation of cell differentiation / positive regulation of ATP biosynthetic process / neuromuscular junction development / Uptake and function of anthrax toxins / positive regulation of peptidyl-serine phosphorylation / MAP kinase kinase kinase activity / response to axon injury / type II interferon-mediated signaling pathway / protein kinase activator activity / ERK1 and ERK2 cascade / Schwann cell development / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / negative regulation of protein-containing complex assembly / response to muscle stretch / neuron projection morphogenesis / myelination / insulin-like growth factor receptor signaling pathway / protein serine/threonine/tyrosine kinase activity / thymus development / CD209 (DC-SIGN) signaling / positive regulation of autophagy / dendrite cytoplasm / response to glucocorticoid / adenylate cyclase activator activity / Signal transduction by L1 / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / wound healing / positive regulation of transcription elongation by RNA polymerase II / RAF activation / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / cellular senescence / small GTPase binding / Stimuli-sensing channels / chemotaxis / neuron differentiation / 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 / Negative regulation of MAPK pathway / insulin receptor signaling pathway / Signaling by BRAF and RAF1 fusions / late endosome / MAPK cascade / protein tyrosine kinase activity / response to oxidative stress / scaffold protein binding / regulation of apoptotic process / cell cortex / microtubule / perikaryon / protein phosphorylation / positive regulation of MAPK cascade / early endosome / positive regulation of ERK1 and ERK2 cascade / protein kinase activity / mitochondrial outer membrane / non-specific serine/threonine protein kinase / ciliary basal body / postsynaptic density
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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Chem-LCJ / RAF proto-oncogene serine/threonine-protein kinase / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsJang, D.M. / Jeon, H. / Eck, M.J.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 10, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Sep 10, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Sep 10, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 8, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RAF proto-oncogene serine/threonine-protein kinase
B: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,3677
Polymers122,8162
Non-polymers1,5515
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 76881.609 Da / Num. of mol.: 1 / Mutation: Q156R, Y340D, Y341D, D587E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P04049, non-specific serine/threonine protein kinase
#2: Protein Dual specificity mitogen-activated protein kinase kinase 1 / MKK1 / ERK activator kinase 1 / MAPK/ERK kinase 1 / MEK 1


Mass: 45934.543 Da / Num. of mol.: 1 / Mutation: S218A, S222A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q02750, mitogen-activated protein kinase kinase
#3: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-LCJ / 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide


Mass: 456.210 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H14FIN4O3
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1CRAF/MEK1/14-3-3 complexCOMPLEX#1-#20RECOMBINANT
2CRAFCOMPLEX#11RECOMBINANT
3MEK1COMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.175 MDaNO
210.076 MDaNO
310.046 MDaNO
410.055 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Spodoptera frugiperda (fall armyworm)7108
43Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 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
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Nominal magnification: 165000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 51.26 e/Å2 / Film or detector model: FEI FALCON I (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7289
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

SoftwareName: PHENIX / Version: 1.21.1_5286 / Classification: refinement
EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175214 / Details: The final resolution was calculated without a mask / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 152.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00444507
ELECTRON MICROSCOPYf_angle_d0.84446096
ELECTRON MICROSCOPYf_chiral_restr0.0495669
ELECTRON MICROSCOPYf_plane_restr0.0144766
ELECTRON MICROSCOPYf_dihedral_angle_d16.29811706

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