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- PDB-8dgs: Cryo-EM structure of a RAS/RAF complex (state 1) -

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Entry
Database: PDB / ID: 8dgs
TitleCryo-EM structure of a RAS/RAF complex (state 1)
Components
  • 14-3-3 protein zeta
  • Dual specificity mitogen-activated protein kinase kinase 1
  • GTPase KRas isoform X2
  • Serine/threonine-protein kinase B-raf
KeywordsTRANSFERASE / kinase complex
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 / CD4-positive, alpha-beta T cell differentiation / positive regulation of axon regeneration / myeloid progenitor cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / mitogen-activated protein kinase kinase / melanosome transport ...negative regulation of homotypic cell-cell adhesion / regulation of vascular associated smooth muscle contraction / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / CD4-positive, alpha-beta T cell differentiation / positive regulation of axon regeneration / myeloid progenitor cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / mitogen-activated protein kinase kinase / melanosome transport / Golgi inheritance / MAP kinase scaffold activity / Signalling to p38 via RIT and RIN / head morphogenesis / endothelial cell apoptotic process / positive regulation of muscle contraction / ARMS-mediated activation / negative regulation of fibroblast migration / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / positive regulation of axonogenesis / vesicle transport along microtubule / regulation of Golgi inheritance / mitogen-activated protein kinase kinase kinase binding / somatic stem cell population maintenance / regulation of T cell differentiation / face development / triglyceride homeostasis / regulation of early endosome to late endosome transport / thyroid gland development / Negative feedback regulation of MAPK pathway / regulation of stress-activated MAPK cascade / Frs2-mediated activation / stress fiber assembly / MAPK3 (ERK1) activation / ERBB2-ERBB3 signaling pathway / positive regulation of protein serine/threonine kinase activity / MAP kinase kinase activity / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / neuromuscular junction development / positive regulation of ATP biosynthetic process / response to axon injury / Uptake and function of anthrax toxins / synaptic vesicle exocytosis / positive regulation of peptidyl-serine phosphorylation / negative regulation of endothelial cell apoptotic process / MAP kinase kinase kinase activity / ERK1 and ERK2 cascade / centriolar satellite / protein kinase activator activity / Schwann cell development / thymus development / postsynaptic modulation of chemical synaptic transmission / positive regulation of stress fiber assembly / substrate adhesion-dependent cell spreading / myelination / neuron projection morphogenesis / positive regulation of substrate adhesion-dependent cell spreading / insulin-like growth factor receptor signaling pathway / protein serine/threonine/tyrosine kinase activity / T cell differentiation in thymus / animal organ morphogenesis / cellular response to calcium ion / positive regulation of autophagy / response to glucocorticoid / dendrite cytoplasm / Signal transduction by L1 / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / small monomeric GTPase / sperm principal piece / sperm end piece / cellular response to xenobiotic stimulus / positive regulation of transcription elongation by RNA polymerase II / visual learning / RAF activation / Signaling by high-kinase activity BRAF mutants / Spry regulation of FGF signaling / MAP2K and MAPK activation / cellular senescence / long-term synaptic potentiation / small GTPase binding / epidermal growth factor receptor signaling pathway / chemotaxis / neuron differentiation / sperm midpiece / 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 / late endosome / regulation of cell population proliferation / T cell receptor signaling pathway / presynapse
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 / Small GTPase Rab domain profile. / Small GTPase Rho domain profile. / Phorbol esters/diacylglycerol binding domain (C1 domain) / : ...: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Small GTPase Rab domain profile. / Small GTPase Rho domain profile. / 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 / Small GTPase, Ras-type / Small GTPase Ras domain profile. / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Small GTP-binding protein domain / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / Chem-LCJ / small monomeric GTPase / Serine/threonine-protein kinase B-raf / Dual specificity mitogen-activated protein kinase kinase 1 / 14-3-3 protein zeta
Similarity search - Component
Biological speciesHomo sapiens (human)
Spodoptera exigua (beet armyworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsEck, M.J. / Jeon, H. / Park, E. / Rawson, S.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P50CA165962 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R35CA242461 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R50CA220830 United States
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM structure of a RAS/RAF recruitment complex.
Authors: Eunyoung Park / Shaun Rawson / Anna Schmoker / Byeong-Won Kim / Sehee Oh / Kangkang Song / Hyesung Jeon / Michael J Eck /
Abstract: RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF ...RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.
History
DepositionJun 24, 2022Deposition site: RCSB / Processing site: RCSB
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Revision 1.1Aug 9, 2023Group: Database references / Category: citation / citation_author
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Revision 1.2Dec 25, 2024Group: Advisory / Data collection ...Advisory / Data collection / Derived calculations / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine/threonine-protein kinase B-raf
B: Dual specificity mitogen-activated protein kinase kinase 1
C: 14-3-3 protein zeta
D: 14-3-3 protein zeta
E: GTPase KRas isoform X2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,52813
Polymers213,3245
Non-polymers2,2048
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 5 molecules ABCDE

#1: Protein Serine/threonine-protein kinase B-raf / Proto-oncogene B-Raf / p94 / v-Raf murine sarcoma viral oncogene homolog B1


Mass: 89402.789 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: ATPgS / Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P15056, 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: 45835.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GDC-0623, ATPgS / 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: Protein 14-3-3 protein zeta


Mass: 28108.514 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera exigua (beet armyworm) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: V9P4T4
#4: Protein GTPase KRas isoform X2


Mass: 21868.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GMPPNP / Source: (gene. exp.) Homo sapiens (human) / Gene: KRAS / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6P5IP77

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Non-polymers , 5 types, 8 molecules

#5: 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
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#8: 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
#9: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3 / Feature type: SUBJECT OF INVESTIGATION
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM

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Details

Has ligand of interestY
Has protein modificationY

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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

ComponentName: Kinase Complex state-1 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.225 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Spodoptera exigua (beet armyworm)7107
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
31Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2800 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 45.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69377 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311306
ELECTRON MICROSCOPYf_angle_d0.44315295
ELECTRON MICROSCOPYf_dihedral_angle_d10.5924304
ELECTRON MICROSCOPYf_chiral_restr0.0381697
ELECTRON MICROSCOPYf_plane_restr0.0031950

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