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- PDB-8vyr: Cryo-EM Structure of the BRAF V600E monomer bound to GDC0879 -

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Basic information

Entry
Database: PDB / ID: 8vyr
TitleCryo-EM Structure of the BRAF V600E monomer bound to GDC0879
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 / 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 / response to cAMP / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / positive regulation of peptidyl-serine phosphorylation / 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 / 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 / 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 / regulation of protein stability / visual learning / response to peptide hormone / centriolar satellite / long-term synaptic potentiation / epidermal growth factor receptor signaling pathway / 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 / Signaling by BRAF and RAF1 fusions / MAPK cascade / 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 / vesicle / blood microparticle
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: 4.32 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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


Theoretical massNumber of molelcules
Total (without water)140,3393
Polymers140,3393
Non-polymers00
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: 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
#2: 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
Has ligand of interestN
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 GDC-0879 / Type: COMPLEX / Entity ID: all / 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 mMGDC-0879C19H18N4O1
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: 4600 nm / Nominal defocus min: 1700 nm
Image recordingElectron dose: 51.55 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: 4.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32966
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: 165.84 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0054802
ELECTRON MICROSCOPYf_angle_d0.97296491
ELECTRON MICROSCOPYf_chiral_restr0.0495759
ELECTRON MICROSCOPYf_plane_restr0.0089856
ELECTRON MICROSCOPYf_dihedral_angle_d12.8271654

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