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- PDB-6q0j: Structure of a MAPK pathway complex -

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Basic information

Entry
Database: PDB / ID: 6q0j
TitleStructure of a MAPK pathway complex
Components
  • 14-3-3 protein zeta
  • Dual specificity mitogen-activated protein kinase kinase 1
  • Serine/threonine-protein kinase B-raf
KeywordsTransferase/PROTEIN BINDING / TRANSFERASE / Transferase-PROTEIN BINDING complex
Function / homology
Function and homology information


MAPK3 (ERK1) activation / Frs2-mediated activation / Spry regulation of FGF signaling / Signalling to p38 via RIT and RIN / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling by BRAF and RAF fusions / Signaling by RAS mutants / Signaling by high-kinase activity BRAF mutants / ARMS-mediated activation / Signaling by moderate kinase activity BRAF mutants ...MAPK3 (ERK1) activation / Frs2-mediated activation / Spry regulation of FGF signaling / Signalling to p38 via RIT and RIN / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling by BRAF and RAF fusions / Signaling by RAS mutants / Signaling by high-kinase activity BRAF mutants / ARMS-mediated activation / Signaling by moderate kinase activity BRAF mutants / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Negative regulation of MAPK pathway / Negative feedback regulation of MAPK pathway / MAP2K and MAPK activation / RAF activation / Uptake and function of anthrax toxins / Signal transduction by L1 / epithelial cell proliferation involved in lung morphogenesis / labyrinthine layer development / mitogen-activated protein kinase kinase / placenta blood vessel development / trehalose metabolism in response to stress / regulation of axon regeneration / cerebellar cortex formation / positive regulation of glucose transmembrane transport / regulation of protein heterodimerization activity / establishment of protein localization to membrane / regulation of Golgi inheritance / trachea formation / face development / regulation of early endosome to late endosome transport / regulation of stress-activated MAPK cascade / positive regulation of axonogenesis / thyroid gland development / cellular senescence / Bergmann glial cell differentiation / signal transduction by protein phosphorylation / positive regulation of production of miRNAs involved in gene silencing by miRNA / cell motility / protein serine/threonine kinase activator activity / activation of MAPKK activity / MAP kinase kinase activity / ERK1 and ERK2 cascade / keratinocyte differentiation / positive regulation of protein serine/threonine kinase activity / cellular response to calcium ion / activation of protein kinase activity / thymus development / neuron differentiation / protein serine/threonine/tyrosine kinase activity / microtubule organizing center / animal organ morphogenesis / cell cycle arrest / scaffold protein binding / chemotaxis / late endosome / heart development / peptidyl-threonine phosphorylation / protein N-terminus binding / positive regulation of peptidyl-serine phosphorylation / protein tyrosine kinase activity / activation of MAPK activity / early endosome / positive regulation of ERK1 and ERK2 cascade / protein C-terminus binding / MAPK cascade / negative regulation of gene expression / non-specific serine/threonine protein kinase / protein kinase activity / intracellular membrane-bounded organelle / negative regulation of cell population proliferation / focal adhesion / protein domain specific binding / protein serine/threonine kinase activity / positive regulation of gene expression / protein phosphorylation / calcium ion binding / protein heterodimerization activity / positive regulation of transcription, DNA-templated / negative regulation of apoptotic process / endoplasmic reticulum / Golgi apparatus / signal transduction / mitochondrion / ATP binding / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm
Serine/Threonine protein kinases active-site signature. / Serine/threonine-protein kinase, active site / Ras-binding domain (RBD) profile. / Zinc finger phorbol-ester/DAG-type profile. / Zinc finger phorbol-ester/DAG-type signature. / 14-3-3 proteins signature 2. / 14-3-3 proteins signature 1. / 14-3-3 protein / Protein kinase domain / Serine-threonine/tyrosine-protein kinase, catalytic domain ...Serine/Threonine protein kinases active-site signature. / Serine/threonine-protein kinase, active site / Ras-binding domain (RBD) profile. / Zinc finger phorbol-ester/DAG-type profile. / Zinc finger phorbol-ester/DAG-type signature. / 14-3-3 proteins signature 2. / 14-3-3 proteins signature 1. / 14-3-3 protein / Protein kinase domain / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / Raf-like Ras-binding / Protein kinase-like domain superfamily / Protein kinase, ATP binding site / Diacylglycerol/phorbol-ester binding / 14-3-3 protein, conserved site / 14-3-3 domain / Ubiquitin-like domain superfamily / 14-3-3 domain superfamily / Protein kinase domain / Phorbol esters/diacylglycerol binding domain (C1 domain) / 14-3-3 protein / Raf-like Ras-binding domain / Protein tyrosine kinase / Protein kinases ATP-binding region signature. / Protein kinase domain profile.
Serine/threonine-protein kinase B-raf / Dual specificity mitogen-activated protein kinase kinase 1 / 14-3-3 protein zeta
Biological speciesHomo sapiens (human)
Spodoptera exigua (beet armyworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 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 InstituteP50CA165962 United States
National Institutes of Health/National Cancer InstituteR50CA221830 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

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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
C: Dual specificity mitogen-activated protein kinase kinase 1
D: Dual specificity mitogen-activated protein kinase kinase 1
X: 14-3-3 protein zeta
Y: 14-3-3 protein zeta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)328,70712
Polymers326,7006
Non-polymers2,0086
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 3 types, 6 molecules ABCDXY

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


Mass: 89306.812 Da / Num. of mol.: 2 / Mutation: S365A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P15056, non-specific serine/threonine protein kinase
#2: Protein/peptide Dual specificity mitogen-activated protein kinase kinase 1 / MKK1 / ERK activator kinase 1 / MAPK/ERK kinase 1 / MEK 1


Mass: 45934.543 Da / Num. of mol.: 2 / Mutation: S218A, S222A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q02750, mitogen-activated protein kinase kinase
#3: Protein/peptide 14-3-3 protein zeta


Mass: 28108.514 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spodoptera exigua (beet armyworm) / References: UniProt: V9P4T4

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Non-polymers , 3 types, 6 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Magnesium
#5: Chemical ChemComp-LCJ / 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide


Mass: 456.210 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C16H14FIN4O3
#6: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S
Comment: ATP-gamma-S (energy-carrying molecule analogue) *YM

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Details

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
Details: insect cell endogenous 14-3-3 co-purified with human BRAF and MEK
Entity ID: 1, 2, 3 / Source: MULTIPLE SOURCES
Molecular weightValue: 325 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
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 425135 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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