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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


CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / Signalling to p38 via RIT and RIN / head morphogenesis / myeloid progenitor cell differentiation / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / endothelial cell apoptotic process ...CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / Signalling to p38 via RIT and RIN / head morphogenesis / myeloid progenitor cell differentiation / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / endothelial cell apoptotic process / negative regulation of fibroblast migration / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / mitogen-activated protein kinase kinase binding / regulation of T cell differentiation / Negative feedback regulation of MAPK pathway / positive regulation of axonogenesis / Frs2-mediated activation / stress fiber assembly / positive regulation of axon regeneration / face development / MAP kinase kinase activity / synaptic vesicle exocytosis / somatic stem cell population maintenance / thyroid gland development / MAP kinase kinase kinase activity / negative regulation of endothelial cell apoptotic process / positive regulation of substrate adhesion-dependent cell spreading / positive regulation of stress fiber assembly / response to cAMP / ERK1 and ERK2 cascade / cellular response to calcium ion / substrate adhesion-dependent cell spreading / thymus development / cellular response to nerve growth factor stimulus / long-term synaptic potentiation / animal organ morphogenesis / RAF activation / Spry regulation of FGF signaling / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / epidermal growth factor receptor signaling pathway / visual learning / response to peptide hormone / 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 / Signaling by BRAF and RAF1 fusions / MAPK cascade / cellular response to xenobiotic stimulus / positive regulation of peptidyl-serine phosphorylation / presynapse / T cell receptor signaling pathway / regulation of cell population proliferation / T cell differentiation in thymus / cell body / scaffold protein binding / negative regulation of neuron apoptotic process / positive regulation of ERK1 and ERK2 cascade / non-specific serine/threonine protein kinase / protein kinase activity / neuron projection / protein phosphorylation / intracellular membrane-bounded organelle / protein serine kinase activity / protein serine/threonine kinase activity / calcium ion binding / protein-containing complex binding / positive regulation of gene expression / negative regulation of apoptotic process / signal transduction / mitochondrion / ATP binding / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
14-3-3 domain / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Delta-Endotoxin; domain 1 / Diacylglycerol/phorbol-ester binding / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. ...14-3-3 domain / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Delta-Endotoxin; domain 1 / 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 / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Ubiquitin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / 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 / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
14-3-3 protein zeta isoform X1 / Serine/threonine-protein kinase B-raf
Similarity search - Component
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.
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
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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: all / 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-HCl1
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 FIELD
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
Refine-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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