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- PDB-7mfe: Autoinhibited BRAF:(14-3-3)2 complex with the BRAF RBD resolved -

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Basic information

Entry
Database: PDB / ID: 7mfe
TitleAutoinhibited BRAF:(14-3-3)2 complex with the BRAF RBD resolved
Components
  • 14-3-3 protein zeta/delta
  • Serine/threonine-protein kinase B-raf
KeywordsSIGNALING PROTEIN / B-Raf / 14-3-3 / B-Raf complex / B-Raf monomer / Inactive B-Raf / Serine/threonine-protein kinase B-raf / RBD
Function / homology
Function and homology information


Golgi reassembly / regulation of synapse maturation / NOTCH4 Activation and Transmission of Signal to the Nucleus / trehalose metabolism in response to stress / CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / establishment of Golgi localization / head morphogenesis / Signalling to p38 via RIT and RIN ...Golgi reassembly / regulation of synapse maturation / NOTCH4 Activation and Transmission of Signal to the Nucleus / trehalose metabolism in response to stress / CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / establishment of Golgi localization / head morphogenesis / Signalling to p38 via RIT and RIN / 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 / Rap1 signalling / negative regulation of fibroblast migration / positive regulation of glucose transmembrane transport / establishment of protein localization to membrane / negative regulation of protein localization to nucleus / mitogen-activated protein kinase kinase binding / regulation of T cell differentiation / Negative feedback regulation of MAPK pathway / KSRP (KHSRP) binds and destabilizes mRNA / GP1b-IX-V activation signalling / positive regulation of axonogenesis / Frs2-mediated activation / stress fiber assembly / positive regulation of axon regeneration / face development / synaptic vesicle exocytosis / somatic stem cell population maintenance / MAP kinase kinase activity / thyroid gland development / Regulation of localization of FOXO transcription factors / Interleukin-3, Interleukin-5 and GM-CSF signaling / MAP kinase kinase kinase activity / phosphoserine residue binding / Activation of BAD and translocation to mitochondria / cellular response to glucose starvation / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / negative regulation of endothelial cell apoptotic process / positive regulation of substrate adhesion-dependent cell spreading / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / response to cAMP / negative regulation of TORC1 signaling / positive regulation of stress fiber assembly / ERK1 and ERK2 cascade / cellular response to calcium ion / negative regulation of innate immune response / substrate adhesion-dependent cell spreading / protein sequestering activity / regulation of ERK1 and ERK2 cascade / cellular response to nerve growth factor stimulus / thymus development / long-term synaptic potentiation / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Deactivation of the beta-catenin transactivating complex / TP53 Regulates Metabolic Genes / Negative regulation of NOTCH4 signaling / animal organ morphogenesis / Spry regulation of FGF signaling / RAF activation / Signaling by high-kinase activity BRAF mutants / visual learning / MAP2K and MAPK activation / epidermal growth factor receptor signaling pathway / 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 / melanosome / MAPK cascade / Signaling by BRAF and RAF1 fusions / cellular response to xenobiotic stimulus / presynapse / cell body / positive regulation of peptidyl-serine phosphorylation / T cell differentiation in thymus / regulation of cell population proliferation / T cell receptor signaling pathway / scaffold protein binding / blood microparticle / DNA-binding transcription factor binding / negative regulation of neuron apoptotic process / vesicle / transmembrane transporter binding / positive regulation of ERK1 and ERK2 cascade / non-specific serine/threonine protein kinase / neuron projection / protein kinase activity / cadherin binding / protein phosphorylation / focal adhesion / protein serine kinase activity / intracellular membrane-bounded organelle
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 / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / 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
Similarity search - Domain/homology
Serine/threonine-protein kinase B-raf / 14-3-3 protein zeta/delta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.07 Å
AuthorsMartinez Fiesco, J.A. / Ping, Z. / Durrant, D.E. / Morrison, D.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 011744 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 010329 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding.
Authors: Juliana A Martinez Fiesco / David E Durrant / Deborah K Morrison / Ping Zhang /
Abstract: RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present ...RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-3:MEK and BRAF:14-3-3 complexes, and an inhibitor-bound, dimeric BRAF:14-3-3 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.
History
DepositionApr 9, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)140,3835
Polymers140,2523
Non-polymers1312
Water0
1


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

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Components

#1: Protein Serine/threonine-protein kinase B-raf / Proto-oncogene B-Raf / p94 / v-Raf murine sarcoma viral oncogene homolog B1


Mass: 84697.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1 / Production host: Homo sapiens (human)
References: UniProt: P15056, non-specific serine/threonine protein kinase
#2: Protein 14-3-3 protein zeta/delta / Protein kinase C inhibitor protein 1 / KCIP-1


Mass: 27777.092 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human) / References: UniProt: P63104
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: Autoinhibited B-Raf:(14-3-3)2 complex with resolved RBD
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMTris1
2200 mMNaClSodium chloride1
310 mMDTT1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 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: 57 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198731 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026961
ELECTRON MICROSCOPYf_angle_d0.5929385
ELECTRON MICROSCOPYf_dihedral_angle_d15.2582612
ELECTRON MICROSCOPYf_chiral_restr0.0391045
ELECTRON MICROSCOPYf_plane_restr0.0041198

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