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- PDB-6q0k: Structure of a MAPK pathway complex -

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Basic information

Entry
Database: PDB / ID: 6q0k
TitleStructure of a MAPK pathway complex
Components
  • 14-3-3 protein zeta/delta
  • Serine/threonine-protein kinase B-raf
KeywordsSIGNALING PROTEIN/Transferase / TRANSFERASE / SIGNALING PROTEIN-Transferase complex
Function / homology
Function and homology information


regulation of synapse maturation / synaptic target recognition / Golgi reassembly / respiratory system process / establishment of Golgi localization / tube formation / dUTP catabolic process / dUMP biosynthetic process / dUTP diphosphatase / dUTP diphosphatase activity ...regulation of synapse maturation / synaptic target recognition / Golgi reassembly / respiratory system process / establishment of Golgi localization / tube formation / dUTP catabolic process / dUMP biosynthetic process / dUTP diphosphatase / dUTP diphosphatase activity / protein targeting / ERK1 and ERK2 cascade / positive regulation of protein insertion into mitochondrial membrane involved in apoptotic signaling pathway / regulation of ERK1 and ERK2 cascade / hippocampal mossy fiber to CA3 synapse / lung development / melanosome / regulation of mRNA stability / platelet activation / membrane organization / ion channel binding / angiogenesis / vesicle / blood microparticle / cadherin binding / glutamatergic synapse / protein domain specific binding / transcription factor binding / cytokine-mediated signaling pathway / focal adhesion / ubiquitin protein ligase binding / protein phosphorylation / protein kinase binding / negative regulation of apoptotic process / signal transduction / magnesium ion binding / mitochondrion / RNA binding / extracellular space / extracellular exosome / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
14-3-3 protein / dUTPase, trimeric / 14-3-3 protein / Deoxyuridine triphosphate nucleotidohydrolase / 14-3-3 protein, conserved site / dUTPase-like / 14-3-3 domain / dUTPase-like superfamily / 14-3-3 domain superfamily / Phorbol esters/diacylglycerol binding domain (C1 domain) ...14-3-3 protein / dUTPase, trimeric / 14-3-3 protein / Deoxyuridine triphosphate nucleotidohydrolase / 14-3-3 protein, conserved site / dUTPase-like / 14-3-3 domain / dUTPase-like superfamily / 14-3-3 domain superfamily / Phorbol esters/diacylglycerol binding domain (C1 domain) / Raf-like Ras-binding domain / Protein tyrosine and serine/threonine kinase
Deoxyuridine 5'-triphosphate nucleotidohydrolase / 14-3-3 protein zeta/delta
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsPark, E. / Rawson, S. / Jeon, H. / Eck, M.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P50CA165962 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R50CA221830 United States
CitationJournal: Nature / Year: 2019
Title: Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.
Authors: Eunyoung Park / Shaun Rawson / Kunhua Li / Byeong-Won Kim / Scott B Ficarro / Gonzalo Gonzalez-Del Pino / Humayun Sharif / Jarrod A Marto / Hyesung Jeon / Michael J Eck /
Abstract: RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival. RAF activity is tightly ...RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 1, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Apr 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Serine/threonine-protein kinase B-raf
B: Serine/threonine-protein kinase B-raf
X: 14-3-3 protein zeta/delta
Y: 14-3-3 protein zeta/delta


Theoretical massNumber of molelcules
Total (without water)234,2004
Polymers234,2004
Non-polymers00
Water0
1


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: 89322.812 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: 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 natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P63104
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: ERK pathway complexMAPK/ERK pathway / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 233 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: OTHER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66215 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-ID
14MNEA1
24MNEB1
33NKXX1
43NKXY1

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