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- PDB-8vyp: Cryo-EM Structure of the BRAF V600E monomer -

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Basic information

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


synaptic target recognition / Golgi reassembly / NOTCH4 Activation and Transmission of Signal to the Nucleus / respiratory system process / establishment of Golgi localization / tube formation / Signalling to p38 via RIT and RIN / ARMS-mediated activation / regulation of synapse maturation / SHOC2 M1731 mutant abolishes MRAS complex function ...synaptic target recognition / Golgi reassembly / NOTCH4 Activation and Transmission of Signal to the Nucleus / respiratory system process / establishment of Golgi localization / tube formation / Signalling to p38 via RIT and RIN / ARMS-mediated activation / regulation of synapse maturation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / negative regulation of protein localization to nucleus / KSRP (KHSRP) binds and destabilizes mRNA / Negative feedback regulation of MAPK pathway / GP1b-IX-V activation signalling / Frs2-mediated activation / MAP kinase kinase activity / lung development / Regulation of localization of FOXO transcription factors / Interleukin-3, Interleukin-5 and GM-CSF signaling / positive regulation of peptidyl-serine phosphorylation / phosphoserine residue binding / Activation of BAD and translocation to mitochondria / regulation of ERK1 and ERK2 cascade / MAP kinase kinase kinase activity / postsynaptic modulation of chemical synaptic transmission / cellular response to glucose starvation / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / negative regulation of TORC1 signaling / animal organ morphogenesis / Transcriptional and post-translational regulation of MITF-M expression and activity / cellular response to calcium ion / negative regulation of innate immune response / hippocampal mossy fiber to CA3 synapse / TP53 Regulates Metabolic Genes / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of protein stability / RAF activation / protein sequestering activity / Signaling by high-kinase activity BRAF mutants / Spry regulation of FGF signaling / Deactivation of the beta-catenin transactivating complex / MAP2K and MAPK activation / Negative regulation of NOTCH4 signaling / epidermal growth factor receptor signaling pathway / intracellular protein localization / 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 / melanosome / angiogenesis / cell body / scaffold protein binding / protein phosphatase binding / blood microparticle / DNA-binding transcription factor binding / vesicle / transmembrane transporter binding / positive regulation of ERK1 and ERK2 cascade / protein kinase activity / protein phosphorylation / non-specific serine/threonine protein kinase / postsynapse / neuron projection / cadherin binding / protein domain specific binding / protein serine kinase activity / focal adhesion / ubiquitin protein ligase binding / protein serine/threonine kinase activity / positive regulation of gene expression / calcium ion binding / negative regulation of apoptotic process / protein kinase binding / negative regulation of transcription by RNA polymerase II / glutamatergic synapse / signal transduction / mitochondrion / : / RNA binding / extracellular exosome / nucleoplasm / zinc ion binding / ATP binding / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
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 / 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. / 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 / 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: 3.29 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: Hugo Lavoie / Ting Jin / Driss Lajoie / Marion Decossas / Patrick Gendron / Bing Wang / Frantisek Filandr / Malha Sahmi / Chang Hwa Jo / Sandra Weber / Geneviève Arseneault / Sasmita ...Authors: Hugo Lavoie / Ting Jin / Driss Lajoie / Marion Decossas / Patrick Gendron / Bing Wang / Frantisek Filandr / Malha Sahmi / Chang Hwa Jo / Sandra Weber / Geneviève Arseneault / Sasmita Tripathy / Pierre Beaulieu / Doris A Schuetz / David C Schriemer / Anne Marinier / William J Rice / Pierre Maisonneuve / Marc Therrien /
Abstract: Uncontrolled activation of the rat sarcoma (RAS)-extracellular signal-regulated kinase (ERK) pathway drives tumor growth, often because of oncogenic BRAF mutations. BRAF regulation, involving ...Uncontrolled activation of the rat sarcoma (RAS)-extracellular signal-regulated kinase (ERK) pathway drives tumor growth, often because of oncogenic BRAF mutations. BRAF regulation, involving monomeric autoinhibition and activation by dimerization, has been intensely scrutinized, but mechanisms enabling oncogenic mutants to evade regulation remain unclear. By using cryo-electron microscopy, we solved the three-dimensional structures of the three oncogenic BRAF mutant classes, including the common V600E variant. These mutations disrupted wild-type BRAF's autoinhibited state, mediated by interactions between the cysteine-rich domain and kinase domain, thereby shifting the kinase domain into a preactivated conformation. This structural change likely results from helix αC displacement. PLX8394, a BRAF inhibitor that stabilizes helix αC in an inactive conformation, restored the autoinhibited conformation of oncogenic BRAF, explaining the properties of this class of compounds.
History
DepositionFeb 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 28, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 11, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jun 25, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 14-3-3 protein zeta/delta
B: 14-3-3 protein zeta/delta
C: Serine/threonine-protein kinase B-raf
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,8624
Polymers140,3393
Non-polymers5231
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 14-3-3 protein zeta/delta / Protein kinase C inhibitor protein 1 / KCIP-1


Mass: 27777.092 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
#2: Protein Serine/threonine-protein kinase B-raf / Proto-oncogene B-Raf / p94 / v-Raf murine sarcoma viral oncogene homolog B1


Mass: 84784.727 Da / Num. of mol.: 1 / Mutation: V600E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: 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
#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.: 1 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
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 / Type: COMPLEX
Details: BRAF V600E - 14-3-3 complex purified by FLAG affinity purification followed by TEV elution from FreeStyle 293-F 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
50.005 mMATPgammaSC10H12Li4N5O12P3S1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / 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: 1900 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 55.64 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117755
Details: Resolution corresponds to the FSC 0.143 cut-off of the masked map
Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 75.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00484501
ELECTRON MICROSCOPYf_angle_d0.57826112
ELECTRON MICROSCOPYf_chiral_restr0.0351730
ELECTRON MICROSCOPYf_plane_restr0.0051767
ELECTRON MICROSCOPYf_dihedral_angle_d5.8585658

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