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- PDB-8chf: cryo-EM Structure of Craf:14-3-3:Mek1 -

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Entry
Database: PDB / ID: 8chf
Titlecryo-EM Structure of Craf:14-3-3:Mek1
Components
  • 14-3-3 protein zeta isoform X1
  • Dual specificity mitogen-activated protein kinase kinase 1
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsSTRUCTURAL PROTEIN / Kinase
Function / homology
Function and homology information


death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / placenta blood vessel development / regulation of axon regeneration / mitogen-activated protein kinase kinase / labyrinthine layer development / type B pancreatic cell proliferation / MAP-kinase scaffold activity ...death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / placenta blood vessel development / regulation of axon regeneration / mitogen-activated protein kinase kinase / labyrinthine layer development / type B pancreatic cell proliferation / MAP-kinase scaffold activity / cerebellar cortex formation / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Signaling by MAP2K mutants / Rap1 signalling / regulation of cell motility / insulin secretion involved in cellular response to glucose stimulus / regulation of Golgi inheritance / trachea formation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / positive regulation of axonogenesis / regulation of stress-activated MAPK cascade / IFNG signaling activates MAPKs / Frs2-mediated activation / GP1b-IX-V activation signalling / ERBB2-ERBB3 signaling pathway / protein kinase activator activity / endodermal cell differentiation / regulation of cell differentiation / face development / MAPK3 (ERK1) activation / pseudopodium / somatic stem cell population maintenance / Bergmann glial cell differentiation / MAP kinase kinase activity / thyroid gland development / neurotrophin TRK receptor signaling pathway / Uptake and function of anthrax toxins / 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 / keratinocyte differentiation / activation of adenylate cyclase activity / response to muscle stretch / ERK1 and ERK2 cascade / myelination / protein serine/threonine/tyrosine kinase activity / CD209 (DC-SIGN) signaling / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / insulin-like growth factor receptor signaling pathway / thymus development / Signal transduction by L1 / cell motility / RAF activation / Signaling by high-kinase activity BRAF mutants / wound healing / MAP2K and MAPK activation / negative regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of protein serine/threonine kinase activity / neuron differentiation / 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 / chemotaxis / MAPK cascade / cellular senescence / Signaling by BRAF and RAF1 fusions / late endosome / insulin receptor signaling pathway / positive regulation of peptidyl-serine phosphorylation / heart development / scaffold protein binding / regulation of apoptotic process / protein tyrosine kinase activity / mitochondrial outer membrane / Ras protein signal transduction / positive regulation of MAPK cascade / positive regulation of ERK1 and ERK2 cascade / early endosome / non-specific serine/threonine protein kinase / protein kinase activity / negative regulation of cell population proliferation / protein phosphorylation / focal adhesion / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / apoptotic process / positive regulation of gene expression / negative regulation of apoptotic process / positive regulation of DNA-templated transcription
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
Chem-29L / 14-3-3 protein zeta isoform X1 / RAF proto-oncogene serine/threonine-protein kinase / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Spodoptera frugiperda (fall armyworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsDedden, D. / Ulrich, G.
Funding support Germany, 1items
OrganizationGrant numberCountry
Other private Germany
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
DepositionFeb 7, 2023Deposition 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.journal_volume / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RAF proto-oncogene serine/threonine-protein kinase
B: RAF proto-oncogene serine/threonine-protein kinase
C: 14-3-3 protein zeta isoform X1
D: 14-3-3 protein zeta isoform X1
E: Dual specificity mitogen-activated protein kinase kinase 1
F: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)290,1798
Polymers289,5106
Non-polymers6692
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12400 Å2
ΔGint-63 kcal/mol
Surface area71730 Å2
MethodPISA

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Components

#1: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 73184.477 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P04049, non-specific serine/threonine protein kinase
#2: Protein 14-3-3 protein zeta isoform X1


Mass: 28108.514 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A9R0D7T1
#3: Protein Dual specificity mitogen-activated protein kinase kinase 1 / MAP kinase kinase 1 / MAPKK 1 / MKK1 / ERK activator kinase 1 / MAPK/ERK kinase 1 / MEK 1


Mass: 43461.938 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q02750, mitogen-activated protein kinase kinase
#4: Chemical ChemComp-29L / 2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridin-4-yl)-1H-pyrazol-1-yl}ethanol


Mass: 334.372 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H18N4O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Complex of dimer phosphorylated C-raf kinase domain with 14-3-3 dimer and 2 subunits of Mek1 containing ligand GDC-0623
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2732
Image scansSampling size: 0.9142 µm / Width: 4096 / Height: 4096

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79383 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementResolution: 4.25→4.25 Å / Cor.coef. Fo:Fc: 0.931 / SU B: 70.636 / SU ML: 0.767 / ESU R: 0.375
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.44498 --
obs0.44498 361986 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 161.235 Å2
Refinement stepCycle: 1 / Total: 12526
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412762
ELECTRON MICROSCOPYf_angle_d0.75317210
ELECTRON MICROSCOPYf_dihedral_angle_d6.1211737
ELECTRON MICROSCOPYf_chiral_restr0.0471903
ELECTRON MICROSCOPYf_plane_restr0.0062194
LS refinement shellResolution: 4.2→4.309 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.607 26993 -
obs--100 %

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