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- PDB-8vys: Cryo-EM Structure of the BRAF V600E monomer bound to PLX8394 -

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Basic information

Entry
Database: PDB / ID: 8vys
TitleCryo-EM Structure of the BRAF V600E monomer bound to PLX8394
Components
  • 14-3-3 protein zeta/delta
  • Serine/threonine-protein kinase B-raf
KeywordsTRANSFERASE/INHIBITOR / BRAF Kinase Monomer Mutant Inhibitor / TRANSFERASE-INHIBITOR complex
Function / homology
Function and homology information


synaptic target recognition / Golgi reassembly / CD4-positive, alpha-beta T cell differentiation / NOTCH4 Activation and Transmission of Signal to the Nucleus / establishment of Golgi localization / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / Signalling to p38 via RIT and RIN / respiratory system process / myeloid progenitor cell differentiation ...synaptic target recognition / Golgi reassembly / CD4-positive, alpha-beta T cell differentiation / NOTCH4 Activation and Transmission of Signal to the Nucleus / establishment of Golgi localization / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / Signalling to p38 via RIT and RIN / respiratory system process / myeloid progenitor cell differentiation / head morphogenesis / regulation of synapse maturation / ARMS-mediated activation / tube formation / endothelial cell apoptotic process / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / negative regulation of fibroblast migration / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / positive regulation of axonogenesis / negative regulation of protein localization to nucleus / regulation of T cell differentiation / mitogen-activated protein kinase kinase binding / Negative feedback regulation of MAPK pathway / KSRP (KHSRP) binds and destabilizes mRNA / GP1b-IX-V activation signalling / Frs2-mediated activation / positive regulation of axon regeneration / stress fiber assembly / face development / MAP kinase kinase activity / synaptic vesicle exocytosis / thyroid gland development / somatic stem cell population maintenance / 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 / postsynaptic modulation of chemical synaptic transmission / negative regulation of endothelial cell apoptotic process / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / protein targeting / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / regulation of ERK1 and ERK2 cascade / cellular response to glucose starvation / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / response to cAMP / RHO GTPases activate PKNs / positive regulation of stress fiber assembly / negative regulation of TORC1 signaling / positive regulation of substrate adhesion-dependent cell spreading / Transcriptional and post-translational regulation of MITF-M expression and activity / ERK1 and ERK2 cascade / substrate adhesion-dependent cell spreading / protein sequestering activity / cellular response to calcium ion / negative regulation of innate immune response / positive regulation of peptidyl-serine phosphorylation / hippocampal mossy fiber to CA3 synapse / thymus development / animal organ morphogenesis / Translocation of SLC2A4 (GLUT4) to the plasma membrane / TP53 Regulates Metabolic Genes / Deactivation of the beta-catenin transactivating complex / lung development / RAF activation / long-term synaptic potentiation / cellular response to nerve growth factor stimulus / Negative regulation of NOTCH4 signaling / Spry regulation of FGF signaling / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / visual learning / regulation of protein stability / epidermal growth factor receptor signaling pathway / response to peptide hormone / Signaling by RAF1 mutants / Negative regulation of MAPK pathway / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / MAPK cascade / Signaling by BRAF and RAF1 fusions / protein localization / melanosome / cellular response to xenobiotic stimulus / presynapse / T cell receptor signaling pathway / regulation of cell population proliferation / T cell differentiation in thymus / cell body / scaffold protein binding / angiogenesis / DNA-binding transcription factor binding / blood microparticle / vesicle / negative regulation of neuron 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. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) ...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 / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Ubiquitin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / 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
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsLavoie, H. / Lajoie, D. / Jin, T. / Decossas, M. / Maisonneuve, P. / Therrien, M.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-388023 Canada
CitationJournal: Science / Year: 2025
Title: BRAF oncogenic mutants evade autoinhibition through a common mechanism
Authors: Lavoie, H. / Jin, T. / Lajoie, D. / Decossas, M. / Gendron, P. / Wang, B. / Filandr, F. / Sahmi, M. / Hwa Jo, C. / Weber, S. / Arseneault, G. / Tripathy, S. / Beaulieu, P. / Schuetz, D.A. / ...Authors: Lavoie, H. / Jin, T. / Lajoie, D. / Decossas, M. / Gendron, P. / Wang, B. / Filandr, F. / Sahmi, M. / Hwa Jo, C. / Weber, S. / Arseneault, G. / Tripathy, S. / Beaulieu, P. / Schuetz, D.A. / Schriemer, D.C. / Marinier, A. / Rice, W.J. / Maisonneuve, P. / Therrien, M.
History
DepositionFeb 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 28, 2025Provider: repository / Type: Initial release

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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: 14-3-3 protein zeta/delta
C: 14-3-3 protein zeta/delta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,6354
Polymers140,0933
Non-polymers5431
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 84800.750 Da / Num. of mol.: 1 / Mutation: V600E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell: FreeStyle 293-F cells / Gene: BRAF, BRAF1, RAFB1 / Plasmid: pCDNA3.1-FLAG-TEV-BRAF V600E / Cell (production host): FreeStyle 293-F cells / Production host: Homo sapiens (human)
References: UniProt: P15056, non-specific serine/threonine protein kinase
#2: Protein 14-3-3 protein zeta/delta / Protein kinase C inhibitor protein 1 / KCIP-1


Mass: 27645.895 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: FreeStyle 293-F cells / References: UniProt: P63104
#3: Chemical ChemComp-A1AEN / (3S)-N-{3-[5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl}-3-fluoropyrrolidine-1-sulfonamide


Mass: 542.533 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H21F3N6O3S / Feature type: SUBJECT OF INVESTIGATION
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: BRAF V600E - 14.3.3 complex bound to PLX8394 / Type: COMPLEX
Details: BRAF complex purified by Flag affinity purification followed by TEV elution from FreeStyle 293F cells
Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.141 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: FreeStyle 293-F cells / Plasmid: pCDNA3.1-FLAG-TEV-BRAF V600E
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H17N2NaO4S1
2137 mMSodium ChlorideNaCl1
35 mMMagnesium chlorideMgCl21
41 mMTCEPC9H15O6P1
50.0005 mMATPgammaSC10H12Li4N5O12P3S1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 80 % / Details: Chameleon system (SPT Labtech)

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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: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 56.44 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4CTFFIND4CTF correction
8PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177000
Details: Resolution corresponds to the FSC 0.143 cut-off of the masked map.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036314
ELECTRON MICROSCOPYf_angle_d0.4548501
ELECTRON MICROSCOPYf_dihedral_angle_d11.0252424
ELECTRON MICROSCOPYf_chiral_restr0.034930
ELECTRON MICROSCOPYf_plane_restr0.0031094

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