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- EMDB-43680: Cryo-EM Structure of the BRAF D594G monomer -

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Basic information

Entry
Database: EMDB / ID: EMD-43680
TitleCryo-EM Structure of the BRAF D594G monomer
Map dataMap of a BRAF D594G - 14-3-3 complex
Sample
  • Complex: BRAF D594G - 14-3-3 complex
    • Protein or peptide: 14-3-3 protein zeta/delta
    • Protein or peptide: Serine/threonine-protein kinase B-raf
KeywordsBRAF 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.76 Å
AuthorsLavoie H / Lajoie D / Jin T / Decossas M / Maisonneuve P / Therrien M
Funding support Canada, 1 items
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, 2024-
Header (metadata) releaseMay 28, 2025-
Map releaseMay 28, 2025-
UpdateJun 11, 2025-
Current statusJun 11, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43680.png

Downloads & links

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Map

FileDownload / File: emd_43680.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of a BRAF D594G - 14-3-3 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.036
Minimum - Maximum-0.091241255 - 0.21674046
Average (Standard dev.)0.00044550118 (±0.007177035)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A of a BRAF D594G - 14-3-3 complex

Fileemd_43680_half_map_1.map
AnnotationHalf map A of a BRAF D594G - 14-3-3 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of a BRAF D594G - 14-3-3 complex

Fileemd_43680_half_map_2.map
AnnotationHalf map B of a BRAF D594G - 14-3-3 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : BRAF D594G - 14-3-3 complex

EntireName: BRAF D594G - 14-3-3 complex
Components
  • Complex: BRAF D594G - 14-3-3 complex
    • Protein or peptide: 14-3-3 protein zeta/delta
    • Protein or peptide: Serine/threonine-protein kinase B-raf

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Supramolecule #1: BRAF D594G - 14-3-3 complex

SupramoleculeName: BRAF D594G - 14-3-3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: BRAF D594G - 14-3-3 complex purified by FLAG affinity purification followed by TEV elution from FreeStyle 293-F cells
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 141 KDa

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Macromolecule #1: 14-3-3 protein zeta/delta

MacromoleculeName: 14-3-3 protein zeta/delta / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.777092 KDa
SequenceString: MDKNELVQKA KLAEQAERYD DMAACMKSVT EQGAELSNEE RNLLSVAYKN VVGARRSSWR VVSSIEQKTE GAEKKQQMAR EYREKIETE LRDICNDVLS LLEKFLIPNA SQAESKVFYL KMKGDYYRYL AEVAAGDDKK GIVDQSQQAY QEAFEISKKE M QPTHPIRL ...String:
MDKNELVQKA KLAEQAERYD DMAACMKSVT EQGAELSNEE RNLLSVAYKN VVGARRSSWR VVSSIEQKTE GAEKKQQMAR EYREKIETE LRDICNDVLS LLEKFLIPNA SQAESKVFYL KMKGDYYRYL AEVAAGDDKK GIVDQSQQAY QEAFEISKKE M QPTHPIRL GLALNFSVFY YEILNSPEKA CSLAKTAFDE AIAELDTLSE ESYKDSTLIM QLLRDNLTLW TSDTQGDEAE AG EGGEN

UniProtKB: 14-3-3 protein zeta/delta

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Macromolecule #2: Serine/threonine-protein kinase B-raf

MacromoleculeName: Serine/threonine-protein kinase B-raf / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human) / Cell: FreeStyle 293-F cells
Molecular weightTheoretical: 42.192016 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: RDRSS(SEP)APNV HINTIEPVNI DDLIRDQGFR GDGGSTTGLS ATPPASLPGS LTNVKALQKS PGPQRERKSS SSSEDR NRM KTLGRRDSSD DWEIPDGQIT VGQRIGSGSF GTVYKGKWHG DVAVKMLNVT APTPQQLQAF KNEVGVLRKT RHVNILL FM GYSTKPQLAI ...String:
RDRSS(SEP)APNV HINTIEPVNI DDLIRDQGFR GDGGSTTGLS ATPPASLPGS LTNVKALQKS PGPQRERKSS SSSEDR NRM KTLGRRDSSD DWEIPDGQIT VGQRIGSGSF GTVYKGKWHG DVAVKMLNVT APTPQQLQAF KNEVGVLRKT RHVNILL FM GYSTKPQLAI VTQWCEGSSL YHHLHIIETK FEMIKLIDIA RQTAQGMDYL HAKSIIHRDL KSNNIFLHED LTVKIGGF G LATVKSRWSG SHQFEQLSGS ILWMAPEVIR MQDKNPYSFQ SDVYAFGIVL YELMTGQLPY SNINNRDQII FMVGRGYLS PDLSKVRSNC PKAMKRLMAE CLKKKRDERP LFPQILASIE LLARSLPKIH RSA(SEP)EPSLN

UniProtKB: Serine/threonine-protein kinase B-raf

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H17N2NaO4SHEPES
137.0 mMNaClSodium Chloride
5.0 mMMgCl2Magnesium Chloride
1.0 mMC9H15O6PTCEP
0.005 mMC10H12Li4N5O12P3SATPgammaS
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Details: Chameleon system (SPT Labtech).

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.55 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.7 µm / Nominal defocus min: 1.7 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.76 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Resolution corresponds to the FSC 0.143 cut-off of the masked map
Number images used: 27791
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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