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- EMDB-16281: Cryo-EM structure of the RAF activating complex KSR-MEK-CNK-HYP -

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Entry
Database: EMDB / ID: EMD-16281
TitleCryo-EM structure of the RAF activating complex KSR-MEK-CNK-HYP
Map dataFinal Map
Sample
  • Complex: Quaternary complex of the kinase domains of KSR and MEK bound to the scaffolding complex CNK-HYP
    • Protein or peptide: Protein aveugle
    • Protein or peptide: Connector enhancer of KSR protein CNK
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase dSOR1
    • Protein or peptide: KSR
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: Trametinib
  • Ligand: MAGNESIUM ION
KeywordsKinase suppressor of Ras (KSR) / Dual specificity mitogen-activated protein kinase kinase (MEK) / Connector enhancer of KSR (CNK) / Protein Aveugle (AVE) / Hyphen protein (HYP) / protein complex / SIGNALING PROTEIN
Function / homology
Function and homology information


hemocyte differentiation / MAPK3 (ERK1) activation / MAPK1 (ERK2) activation / Frs2-mediated activation / Negative feedback regulation of MAPK pathway / terminal region determination / Signal transduction by L1 / RAF activation / Phosphorylation of CI / compound eye cone cell differentiation ...hemocyte differentiation / MAPK3 (ERK1) activation / MAPK1 (ERK2) activation / Frs2-mediated activation / Negative feedback regulation of MAPK pathway / terminal region determination / Signal transduction by L1 / RAF activation / Phosphorylation of CI / compound eye cone cell differentiation / Phosphorylation of SMO / terminal branching, open tracheal system / torso signaling pathway / photoreceptor cell development / tracheal outgrowth, open tracheal system / R7 cell fate commitment / compound eye photoreceptor cell differentiation / sevenless signaling pathway / Phosphorylation of PER and TIM / MAP2K and MAPK activation / eye photoreceptor cell differentiation / epithelial cell migration, open tracheal system / imaginal disc-derived wing vein specification / border follicle cell migration / imaginal disc-derived wing morphogenesis / anterior/posterior axis specification, embryo / cellular response to X-ray / mitogen-activated protein kinase kinase / MAP-kinase scaffold activity / mitogen-activated protein kinase kinase kinase binding / mitotic DNA replication checkpoint signaling / dorsal/ventral pattern formation / positive regulation of Ras protein signal transduction / mitotic G2 DNA damage checkpoint signaling / MAP kinase kinase activity / fibroblast growth factor receptor signaling pathway / enzyme regulator activity / vascular endothelial growth factor receptor signaling pathway / condensed chromosome / ERK1 and ERK2 cascade / visual perception / determination of adult lifespan / epidermal growth factor receptor signaling pathway / cytoplasmic side of plasma membrane / receptor tyrosine kinase binding / kinase binding / cell surface receptor protein tyrosine kinase signaling pathway / MAPK cascade / apical part of cell / insulin receptor signaling pathway / cell cortex / scaffold protein binding / protein tyrosine kinase activity / defense response to virus / Ras protein signal transduction / positive regulation of ERK1 and ERK2 cascade / non-specific serine/threonine protein kinase / protein kinase activity / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / ATP binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Aveugle-like, SAM domain / : / CRIC domain / Connector enhancer of kinase suppressor of ras / CRIC domain profile. / Kinase suppressor of RAS, SAM-like domain / SAM like domain present in kinase suppressor RAS 1 / Kinase suppressor RAS 1, N-terminal helical hairpin / Kinase suppressor RAS 1, N-terminal helical hairpin superfamily / Kinase suppressor RAS 1 N-terminal helical hairpin ...Aveugle-like, SAM domain / : / CRIC domain / Connector enhancer of kinase suppressor of ras / CRIC domain profile. / Kinase suppressor of RAS, SAM-like domain / SAM like domain present in kinase suppressor RAS 1 / Kinase suppressor RAS 1, N-terminal helical hairpin / Kinase suppressor RAS 1, N-terminal helical hairpin superfamily / Kinase suppressor RAS 1 N-terminal helical hairpin / SAM domain (Sterile alpha motif) / 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 / SAM domain (Sterile alpha motif) / C1-like domain superfamily / SAM domain profile. / Sterile alpha motif. / Sterile alpha motif domain / PH domain / Sterile alpha motif/pointed domain superfamily / PH domain profile. / Pleckstrin homology domain. / PDZ domain / Pleckstrin homology domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Tyrosine protein kinases specific active-site signature. / PH-like domain superfamily / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / 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
non-specific serine/threonine protein kinase / Dual specificity mitogen-activated protein kinase kinase dSOR1 / Connector enhancer of KSR protein CNK / Protein aveugle
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsMaisonneuve P / Fronzes R / Sicheri F
Funding support Canada, France, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FRN-414829 Canada
Foundation for Medical Research (France)AJE202110014546 France
Canadian Institutes of Health Research (CIHR)FDN-143277 Canada
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: The CNK-HYP scaffolding complex promotes RAF activation by enhancing KSR-MEK interaction.
Authors: Pierre Maisonneuve / Malha Sahmi / Fanny Bergeron-Labrecque / Xianjie Iris Ma / Juliette Queguiner / Geneviève Arseneault / Martin Lefrançois / Igor Kurinov / Rémi Fronzes / Frank Sicheri / Marc Therrien /
Abstract: The RAS-MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and ...The RAS-MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and the kinases RAF, MEK and ERK. Activation of RAF by RAS is notoriously intricate and remains only partially understood. There are three RAF isoforms in mammals (ARAF, BRAF and CRAF) and two related pseudokinases (KSR1 and KSR2). RAS-mediated activation of RAF depends on an allosteric mechanism driven by the dimerization of its kinase domain. Recent work on human RAFs showed that MEK binding to KSR1 promotes KSR1-BRAF heterodimerization, which leads to the phosphorylation of free MEK molecules by BRAF. Similar findings were made with the single Drosophila RAF homolog. Here we show that the fly scaffold proteins CNK and HYP stabilize the KSR-MEK interaction, which in turn enhances RAF-KSR heterodimerization and RAF activation. The cryogenic electron microscopy structure of the minimal KSR-MEK-CNK-HYP complex reveals a ring-like arrangement of the CNK-HYP complex allowing CNK to simultaneously engage KSR and MEK, thus stabilizing the binary interaction. Together, these results illuminate how CNK contributes to RAF activation by stimulating the allosteric function of KSR and highlight the diversity of mechanisms impacting RAF dimerization as well as the regulatory potential of the KSR-MEK interaction.
History
DepositionDec 6, 2022-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16281.png

Downloads & links

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Map

FileDownload / File: emd_16281.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.93 Å/pix.
x 360 pix.
= 334.8 Å		0.93 Å/pix.
x 360 pix.
= 334.8 Å		0.93 Å/pix.
x 360 pix.
= 334.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.0
Minimum - Maximum-56.064860000000003 - 67.055620000000005
Average (Standard dev.)0.000000000001615 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 334.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16281_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_16281_half_map_1.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_16281_half_map_2.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Quaternary complex of the kinase domains of KSR and MEK bound to ...

EntireName: Quaternary complex of the kinase domains of KSR and MEK bound to the scaffolding complex CNK-HYP
Components
  • Complex: Quaternary complex of the kinase domains of KSR and MEK bound to the scaffolding complex CNK-HYP
    • Protein or peptide: Protein aveugle
    • Protein or peptide: Connector enhancer of KSR protein CNK
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase dSOR1
    • Protein or peptide: KSR
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: Trametinib
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Quaternary complex of the kinase domains of KSR and MEK bound to ...

SupramoleculeName: Quaternary complex of the kinase domains of KSR and MEK bound to the scaffolding complex CNK-HYP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: Mg2+/ANP and Trametinib ligands bound to MEK
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 132 KDa

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Macromolecule #1: Protein aveugle

MacromoleculeName: Protein aveugle / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 13.143911 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GAMDPGEETI NSTQNKTRTK TTRPKAVYLW TVSDVLKWYR RHCGEYTQYE QLFAQHDITG RALLRITDSS LQRMGVTDNR DREAIWREI VKQRLKTDIM EIRDMERLNI Y

UniProtKB: Protein aveugle

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Macromolecule #2: Connector enhancer of KSR protein CNK

MacromoleculeName: Connector enhancer of KSR protein CNK / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 38.534203 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMDPMAYIN IAEWTPDQVT DWIKGLDESM KGYLYEFSKQ EIGGRALLNI RPYELENLGM LRIGHQEIVL EAVENLRNFH YHLKNDNLQ FMALHVATAA KNLHRELARN HAESTKIDTR ILHDITRTIA TLKPLVGSLE RTPFRKQEMY REYCGNVLKC G LELATIAH ...String:
GAMDPMAYIN IAEWTPDQVT DWIKGLDESM KGYLYEFSKQ EIGGRALLNI RPYELENLGM LRIGHQEIVL EAVENLRNFH YHLKNDNLQ FMALHVATAA KNLHRELARN HAESTKIDTR ILHDITRTIA TLKPLVGSLE RTPFRKQEMY REYCGNVLKC G LELATIAH RDRFALQPVP AIRQSAERLE NLANFVIQDI SDPMVLQPAS LNLVTLKKRE SELGFNIESS YNGIHRVTDI KY NSPAHNS GKIEDGDEIV QINYQTVVGW QHRTVLEHLR EALPDVVLTV KKRPKHTKMF GQIYMQPYRL PSKKRNMAAR WAA QMPSPR AAFLTLDTE

UniProtKB: Connector enhancer of KSR protein CNK

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Macromolecule #3: Dual specificity mitogen-activated protein kinase kinase dSOR1

MacromoleculeName: Dual specificity mitogen-activated protein kinase kinase dSOR1
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: mitogen-activated protein kinase kinase
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 43.926496 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSKNKLNLVL PPVNTEATVA AATVAPTPPF KTPSGTDTHS LLGKPKTSID ALTETLEGLD MGDTERKRIK MFLSQKEKIG ELSDEDLEK LGELGSGNGG VVMKVRHTHT HLIMARKLIH LEVKPAIKKQ ILRELKVLHE CNFPHIVGFY GAFYSDGEIS I CMEYMDGG ...String:
MSKNKLNLVL PPVNTEATVA AATVAPTPPF KTPSGTDTHS LLGKPKTSID ALTETLEGLD MGDTERKRIK MFLSQKEKIG ELSDEDLEK LGELGSGNGG VVMKVRHTHT HLIMARKLIH LEVKPAIKKQ ILRELKVLHE CNFPHIVGFY GAFYSDGEIS I CMEYMDGG SLDLILKRAG RIPESILGRI TLAVLKGLSY LRDNHAIIHR DVKPSNILVN SSGEIKICDF GVSGQLIDSM AN SFVGTRS YMSPERLQGT HYSVQSDIWS LGLSLVEMAI GMYPIPPPNT ATLESIFADN AEESGQPTDE PRAMAIFELL DYI VNEPPP KLEHKIFSTE FKDFVDICLK KQPDERADLK TLLSHPWIRK AELEEVDISG WVCKTMDLPP STPKRNTSPN

UniProtKB: Dual specificity mitogen-activated protein kinase kinase dSOR1

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Macromolecule #4: KSR

MacromoleculeName: KSR / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 36.474898 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GGRTEDGDSG QWRQNSISLK EWDIPYGDLL LLERIGQGRF GTVHRALWHG DVAVKLLNED YLQDEHMLET FRSEVANFKN TRHENLVLF MGACMNPPYL AIVTSLCKGN TLYTYIHQRR EKFAMNRTLL IAQQIAQGMG YLHAREIIHK DLRTKNIFIE N GKVIITDF ...String:
GGRTEDGDSG QWRQNSISLK EWDIPYGDLL LLERIGQGRF GTVHRALWHG DVAVKLLNED YLQDEHMLET FRSEVANFKN TRHENLVLF MGACMNPPYL AIVTSLCKGN TLYTYIHQRR EKFAMNRTLL IAQQIAQGMG YLHAREIIHK DLRTKNIFIE N GKVIITDF GLFSSTKLLY CDMGLGVPHN WLCYLAPELI RALQPEKPRG ECLEFTPYSD VYSFGTVWYE LICGEFTFKD QP AESIIWQ VGRGMKQSLA NLQSGRDVKD LLMLCWTYEK EHRPQFARLL SLLEHLPKKR LARSPSHPVN LSRSAESVF

UniProtKB: non-specific serine/threonine protein kinase

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #6: Trametinib

MacromoleculeName: Trametinib / type: ligand / ID: 6 / Number of copies: 1 / Formula: QOM
Molecular weightTheoretical: 615.395 Da
Chemical component information

ChemComp-QOM:
Trametinib / medication, anticancer, inhibitor*YM / Trametinib

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.79 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMHEPESN-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
500.0 mMNaClSodium chlorideSodium Chloride
1.0 mMTCEPTris(2-carboxyethyl)phosphine hydrochloride
25.0 mMMgCl2Magnesium Chloride

Details: AMPPNP 25mM Trametinib 0.05mM
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.00026000000000000003 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: 3.5 sec blot time 1 sec drain time.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 26.398 µm / Nominal defocus min: 5.212 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 44 / Average electron dose: 1.15 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

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

7jur

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 141531

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