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- PDB-8cpd: Cryo-EM structure of CRaf dimer with 14:3:3 -

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Basic information

Entry
Database: PDB / ID: 8cpd
TitleCryo-EM structure of CRaf dimer with 14:3:3
Components
  • 14-3-3 protein zeta isoform X1
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsCYTOKINE / Kinase / Complex
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 / wound healing / MAP2K and MAPK activation / 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 / Ras protein signal transduction / 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 / nucleus / metal ion binding / plasma membrane / cytoplasm / cytosol
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsDedden, D. / Graedler, U. / Schwarz, D. / Thomsen, M. / Leuthner, B. / Schneider, E. / Nitsche, J.
Funding support1items
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, 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
C: 14-3-3 protein zeta isoform X1
B: RAF proto-oncogene serine/threonine-protein kinase
D: 14-3-3 protein zeta isoform X1


Theoretical massNumber of molelcules
Total (without water)202,5864
Polymers202,5864
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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
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: Tetrameric complex of phosphorylated CRAF dimer with 14-3-3 isoform zeta
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 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: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 653449 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048326
ELECTRON MICROSCOPYf_angle_d0.71211236
ELECTRON MICROSCOPYf_dihedral_angle_d4.5711112
ELECTRON MICROSCOPYf_chiral_restr0.0421252
ELECTRON MICROSCOPYf_plane_restr0.0061430

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