[English] 日本語
Yorodumi
- PDB-8bw9: Cryo-EM structure of the RAF activating complex KSR-MEK-CNK-HYP -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8bw9
TitleCryo-EM structure of the RAF activating complex KSR-MEK-CNK-HYP
Components
  • Connector enhancer of KSR protein CNK
  • Dual specificity mitogen-activated protein kinase kinase dSOR1
  • KSR
  • Protein aveugle
KeywordsSIGNALING PROTEIN / Kinase 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
Function / homology
Function and homology information


hemocyte differentiation / MAPK3 (ERK1) activation / MAPK1 (ERK2) activation / Frs2-mediated activation / Signal transduction by L1 / Negative feedback regulation of MAPK pathway / terminal region determination / RAF activation / Phosphorylation of CI / compound eye cone cell differentiation ...hemocyte differentiation / MAPK3 (ERK1) activation / MAPK1 (ERK2) activation / Frs2-mediated activation / Signal transduction by L1 / Negative feedback regulation of MAPK pathway / terminal region determination / 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 / vascular endothelial growth factor receptor signaling pathway / enzyme regulator activity / condensed chromosome / cell surface receptor protein tyrosine kinase signaling pathway / visual perception / ERK1 and ERK2 cascade / determination of adult lifespan / epidermal growth factor receptor signaling pathway / cytoplasmic side of plasma membrane / receptor tyrosine kinase binding / kinase binding / MAPK cascade / insulin receptor signaling pathway / apical part of cell / 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 / cytoplasm / cytosol
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. / PDZ domain / Pleckstrin homology 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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Trametinib / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsMaisonneuve, P. / Fronzes, R. / Sicheri, F.
Funding support Canada, France, 3items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN
Revision 1.2Mar 6, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Protein aveugle
B: Connector enhancer of KSR protein CNK
C: Dual specificity mitogen-activated protein kinase kinase dSOR1
D: KSR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,2257
Polymers132,0804
Non-polymers1,1463
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9690 Å2
ΔGint-57 kcal/mol
Surface area43810 Å2
MethodPISA

-
Components

-
Protein , 4 types, 4 molecules ABCD

#1: Protein Protein aveugle


Mass: 13143.911 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: ave, CG30476 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8ML92
#2: Protein Connector enhancer of KSR protein CNK / Connector enhancer of ksr / isoform A / isoform B / SD09985p


Mass: 38534.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: cnk, anon-WO0140519.129, anon-WO0257455.27, CNK, Cnk, Dmel\CG6556, EC2-3, EK2-3, l(2)k16314, sag, CG6556, Dmel_CG6556
Production host: Escherichia coli (E. coli) / References: UniProt: Q7KNQ9
#3: Protein Dual specificity mitogen-activated protein kinase kinase dSOR1 / Downstream of RAF / MAPKK


Mass: 43926.496 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Dsor1, CG15793 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q24324, mitogen-activated protein kinase kinase
#4: Protein KSR


Mass: 36474.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: ksr, CG2899 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q24170

-
Non-polymers , 3 types, 3 molecules

#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-QOM / Trametinib / N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide


Mass: 615.395 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H23FIN5O4 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication, anticancer, inhibitor*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

-
Details

Has ligand of interestY

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

ComponentName: Quaternary complex of the kinase domains of KSR and MEK bound to the scaffolding complex CNK-HYP
Type: COMPLEX / Details: Mg2+/ANP and Trametinib ligands bound to MEK / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.132 MDa / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7 / Details: AMPPNP 25mM Trametinib 0.05mM
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMN-2-hydroxyethylpiperazine-N-2-ethane sulfonic acidHEPES1
2500 mMSodium ChlorideNaCl1
31 mMTris(2-carboxyethyl)phosphine hydrochlorideTCEP1
425 mMMagnesium ChlorideMgCl21
SpecimenConc.: 0.79 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: 3.5 sec blot time 1 sec drain time

-
Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 26398 nm / Nominal defocus min: 5212 nm
Image recordingElectron dose: 1.15 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 44

-
Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141531 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 119.78 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037105
ELECTRON MICROSCOPYf_angle_d0.56329619
ELECTRON MICROSCOPYf_chiral_restr0.0421073
ELECTRON MICROSCOPYf_plane_restr0.0051255
ELECTRON MICROSCOPYf_dihedral_angle_d4.2591993

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbjlvh1.pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more