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- EMDB-16779: Cryo-EM structure of CRaf dimer with 14:3:3 -

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Basic information

Entry
Database: EMDB / ID: EMD-16779
TitleCryo-EM structure of CRaf dimer with 14:3:3
Map data
Sample
  • Complex: Tetrameric complex of phosphorylated CRAF dimer with 14-3-3 isoform zeta
    • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Protein or peptide: 14-3-3 protein zeta isoform X1
KeywordsKinase / Complex / CYTOKINE
Function / homology
Function and homology information


death-inducing signaling complex assembly / intermediate filament cytoskeleton organization / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / regulation of cell motility / insulin secretion involved in cellular response to glucose stimulus / Negative feedback regulation of MAPK pathway ...death-inducing signaling complex assembly / intermediate filament cytoskeleton organization / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / regulation of cell motility / insulin secretion involved in cellular response to glucose stimulus / Negative feedback regulation of MAPK pathway / IFNG signaling activates MAPKs / GP1b-IX-V activation signalling / ERBB2-ERBB3 signaling pathway / regulation of cell differentiation / face development / pseudopodium / somatic stem cell population maintenance / thyroid gland development / neurotrophin TRK receptor signaling pathway / extrinsic apoptotic signaling pathway via death domain receptors / MAP kinase kinase kinase activity / negative regulation of protein-containing complex assembly / Schwann cell development / type II interferon-mediated signaling pathway / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / activation of adenylate cyclase activity / response to muscle stretch / myelination / CD209 (DC-SIGN) signaling / insulin-like growth factor receptor signaling pathway / thymus development / RAF activation / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / wound healing / negative regulation of cysteine-type endopeptidase activity involved in apoptotic process / Stimuli-sensing channels / Negative regulation of MAPK pathway / 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 / Signaling by BRAF and RAF1 fusions / insulin receptor signaling pathway / positive regulation of peptidyl-serine phosphorylation / regulation of apoptotic process / mitochondrial outer membrane / positive regulation of MAPK cascade / non-specific serine/threonine protein kinase / protein kinase activity / negative regulation of cell population proliferation / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / apoptotic process / negative regulation of apoptotic process / Golgi apparatus / enzyme binding / signal transduction / positive regulation of transcription by RNA polymerase II / mitochondrion / ATP binding / identical protein binding / metal ion 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) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain ...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 / 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
Similarity search - Domain/homology
14-3-3 protein zeta isoform X1 / RAF proto-oncogene serine/threonine-protein kinase
Similarity search - Component
Biological speciesHomo sapiens (human) / Spodoptera frugiperda (fall armyworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsDedden D / Graedler U / Schwarz D / Thomsen M / Leuthner B / Schneider E / Nitsche J
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: J Mol Biol / Year: 2024
Title: Cryo-EM Structures of CRAF/14-3-3 and CRAF/14-3-3/MEK1 Complexes.
Authors: Dirk Dedden / Julius Nitsche / Elisabeth V Schneider / Maren Thomsen / Daniel Schwarz / Birgitta Leuthner / Ulrich Grädler /
Abstract: RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) ...RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF/14-3-3 at 3.4 Å resolution and CRAF/14-3-3/MEK1 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF/14-3-3 and CRAF/14-3-3/MEK1 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF/14-3-3/MEK1 complex.
History
DepositionMar 2, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16779.png

Downloads & links

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Map

FileDownload / File: emd_16779.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.731 Å
Density
Contour LevelBy AUTHOR: 0.153
Minimum - Maximum-0.50221443 - 0.7062712
Average (Standard dev.)0.0013063657 (±0.020929947)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 187.136 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16779_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16779_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrameric complex of phosphorylated CRAF dimer with 14-3-3 isofo...

EntireName: Tetrameric complex of phosphorylated CRAF dimer with 14-3-3 isoform zeta
Components
  • Complex: Tetrameric complex of phosphorylated CRAF dimer with 14-3-3 isoform zeta
    • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Protein or peptide: 14-3-3 protein zeta isoform X1

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Supramolecule #1: Tetrameric complex of phosphorylated CRAF dimer with 14-3-3 isofo...

SupramoleculeName: Tetrameric complex of phosphorylated CRAF dimer with 14-3-3 isoform zeta
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: RAF proto-oncogene serine/threonine-protein kinase

MacromoleculeName: RAF proto-oncogene serine/threonine-protein kinase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73.184477 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEHIQGAWKT ISNGFGFKDA VFDGSSCISP TIVQQFGYQR RASDDGKLTD PSKTSNTIRV FLPNKQRTVV NVRNGMSLHD CLMKALKVR GLQPECCAVF RLLHEHKGKK ARLDWNTDAA SLIGEELQVD FLDHVPLTTH NFARKTFLKL AFCDICQKFL L NGFRCQTC ...String:
MEHIQGAWKT ISNGFGFKDA VFDGSSCISP TIVQQFGYQR RASDDGKLTD PSKTSNTIRV FLPNKQRTVV NVRNGMSLHD CLMKALKVR GLQPECCAVF RLLHEHKGKK ARLDWNTDAA SLIGEELQVD FLDHVPLTTH NFARKTFLKL AFCDICQKFL L NGFRCQTC GYKFHEHCST KVPTMCVDWS NIRQLLLFPN STIGDSGVPA LPSLTMRRMR ESVSRMPVSS QHRYSTPHAF TF NTSSPSS EGSLSQRQRS TSTPNVHMVS TTLPVDSRMI EDAIRSHSES ASPSALSSSP NNLSPTGWSQ PKTPVPAQRE RAP VSGTQE KNKIRPRGQR DSSYDWEIEA SEVMLSTRIG SGSFGTVYKG KWHGDVAVKI LKVVDPTPEQ FQAFRNEVAV LRKT RHVNI LLFMGYMTKD NLAIVTQWCE GSSLYKHLHV QETKFQMFQL IDIARQTAQG MDYLHAKNII HRDMKSNNIF LHEGL TVKI GDFGLATVKS RWSGSQQVEQ PTGSVLWMAP EVIRMQDNNP FSFQSDVYSY GIVLYELMTG ELPYSHINNR DQIIFM VGR GYASPDLSKL YKNCPKAMKR LVADCVKKVK EERPLFPQIL SSIELLQHSL PKINRSA(SEP)EP SLHRAAHTED INA CTLTTS PRLPVF

UniProtKB: RAF proto-oncogene serine/threonine-protein kinase

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Macromolecule #2: 14-3-3 protein zeta isoform X1

MacromoleculeName: 14-3-3 protein zeta isoform X1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 28.108514 KDa
SequenceString: MSVDKEELVQ RAKLAEQAER YDDMAAAMKE VTETGVELSN EERNLLSVAY KNVVGARRSS WRVISSIEQK TEGSERKQQM AKEYRVKVE KELREICYDV LGLLDKHLIP KASNPESKVF YLKMKGDYYR YLAEVATGET RNSVVEDSQK AYQDAFEISK A KMQPTHPI ...String:
MSVDKEELVQ RAKLAEQAER YDDMAAAMKE VTETGVELSN EERNLLSVAY KNVVGARRSS WRVISSIEQK TEGSERKQQM AKEYRVKVE KELREICYDV LGLLDKHLIP KASNPESKVF YLKMKGDYYR YLAEVATGET RNSVVEDSQK AYQDAFEISK A KMQPTHPI RLGLALNFSV FYYEILNSPD KACQLAKQAF DDAIAELDTL NEDSYKDSTL IMQLLRDNLT LWTSDTQGDG DE PAEGGDN

UniProtKB: 14-3-3 protein zeta isoform X1

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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

Concentration0.3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 65.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3omv

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 653449

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