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- PDB-6uan: B-Raf:14-3-3 complex -

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Basic information

Entry
Database: PDB / ID: 6uan
TitleB-Raf:14-3-3 complex
Components
  • 14-3-3 zeta
  • Serine/threonine-protein kinase B-raf
KeywordsSIGNALING PROTEIN / Complex Kinase
Function / homology
Function and homology information


trehalose metabolism in response to stress / positive regulation of glucose transmembrane transport / establishment of protein localization to membrane / activation of MAPKK activity / cellular response to calcium ion / animal organ morphogenesis / epidermal growth factor receptor signaling pathway / scaffold protein binding / positive regulation of peptidyl-serine phosphorylation / positive regulation of ERK1 and ERK2 cascade ...trehalose metabolism in response to stress / positive regulation of glucose transmembrane transport / establishment of protein localization to membrane / activation of MAPKK activity / cellular response to calcium ion / animal organ morphogenesis / epidermal growth factor receptor signaling pathway / scaffold protein binding / positive regulation of peptidyl-serine phosphorylation / positive regulation of ERK1 and ERK2 cascade / MAPK cascade / non-specific serine/threonine protein kinase / protein kinase activity / intracellular membrane-bounded organelle / protein serine/threonine kinase activity / protein phosphorylation / positive regulation of gene expression / calcium ion binding / negative regulation of apoptotic process / mitochondrion / ATP binding / identical protein binding / plasma membrane / nucleus / cytosol
14-3-3 protein / 14-3-3 protein, conserved site / Raf-like Ras-binding / Serine/threonine-protein kinase, active site / Protein kinase-like domain superfamily / Protein kinase, ATP binding site / Diacylglycerol/phorbol-ester binding / Protein kinase domain / 14-3-3 domain / Ubiquitin-like domain superfamily ...14-3-3 protein / 14-3-3 protein, conserved site / Raf-like Ras-binding / Serine/threonine-protein kinase, active site / Protein kinase-like domain superfamily / Protein kinase, ATP binding site / Diacylglycerol/phorbol-ester binding / Protein kinase domain / 14-3-3 domain / Ubiquitin-like domain superfamily / 14-3-3 domain superfamily / 14-3-3 protein / Phorbol esters/diacylglycerol binding domain (C1 domain) / Raf-like Ras-binding domain / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / 14-3-3 domain / Delta-Endotoxin; domain 1 / Up-down Bundle / Mainly Alpha
14-3-3 zeta / Serine/threonine-protein kinase B-raf
Biological speciesHomo sapiens (human)
Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKondo, Y. / Ognjenovic, J. / Banerjee, S. / Karandur, D. / Merk, A. / Kulhanek, K. / Wong, K. / Roose, J.P. / Subramaniam, S. / Kuriyan, J.
Funding support United States, Canada, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P01-AI091580 United States
Canada Excellence Research Chair Award Canada
Citation
Journal: Science / Year: 2019
Title: Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases.
Authors: Yasushi Kondo / Jana Ognjenović / Saikat Banerjee / Deepti Karandur / Alan Merk / Kayla Kulhanek / Kathryn Wong / Jeroen P Roose / Sriram Subramaniam / John Kuriyan /
Abstract: Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo-electron microscopy structural analysis of a phosphorylated B-Raf kinase ...Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo-electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of ~3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical "active" conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.
#1: Journal: Science / Year: 2019
Title: Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases
Authors: Kondo, Y. / Ognjenovic, J. / Banerjee, S. / Karandur, D. / Merk, A. / Kulhanek, K. / Wong, K. / Roose, J.P. / Subramaniam, S. / Kuriyan, J.
Validation Report
SummaryFull reportAbout validation report
History
DepositionSep 11, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / Database references / Category: citation / citation_author
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: 14-3-3 zeta
Z: 14-3-3 zeta
B: Serine/threonine-protein kinase B-raf
C: Serine/threonine-protein kinase B-raf


Theoretical massNumber of molelcules
Total (without water)225,7814
Polymers225,7814
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 14-3-3 zeta


Mass: 28108.514 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: A0A068JLL8
#2: Protein Serine/threonine-protein kinase B-raf / Proto-oncogene B-Raf / p94 / v-Raf murine sarcoma viral oncogene homolog B1


Mass: 84781.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: P15056, non-specific serine/threonine protein kinase
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: Ternary complex of B-Raf kinase domain dimer and 14-3-3 dimer
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMTris-HClTris1
2150 mMsodium chloride1
30.5 mMTCEP1
SpecimenConc.: 0.2 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 R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278 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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113313 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0077828
ELECTRON MICROSCOPYf_angle_d0.92310616
ELECTRON MICROSCOPYf_dihedral_angle_d7.7654743
ELECTRON MICROSCOPYf_chiral_restr0.0561218
ELECTRON MICROSCOPYf_plane_restr0.0061358

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