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

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Basic information

Entry
Database: PDB / ID: 6nyb
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
Function / homology
Function and homology information


Spry regulation of FGF signaling / MAPK3 (ERK1) activation / Signalling to p38 via RIT and RIN / ARMS-mediated activation / Signal transduction by L1 / Uptake and function of anthrax toxins / RAF activation / MAP2K and MAPK activation / Negative feedback regulation of MAPK pathway / Negative regulation of MAPK pathway ...Spry regulation of FGF signaling / MAPK3 (ERK1) activation / Signalling to p38 via RIT and RIN / ARMS-mediated activation / Signal transduction by L1 / Uptake and function of anthrax toxins / RAF activation / MAP2K and MAPK activation / Negative feedback regulation of MAPK pathway / Negative regulation of MAPK pathway / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Signaling by moderate kinase activity BRAF mutants / Frs2-mediated activation / Signaling by high-kinase activity BRAF mutants / Signaling by RAS mutants / Signaling by BRAF and RAF fusions / Paradoxical activation of RAF signaling by kinase inactive BRAF / 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 / establishment of protein localization to membrane / regulation of Golgi inheritance / trachea formation / face development / regulation of early endosome to late endosome transport / positive regulation of axonogenesis / thyroid gland development / regulation of stress-activated MAPK cascade / 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 / stress-activated protein kinase signaling cascade / ERK1 and ERK2 cascade / MAP kinase kinase activity / keratinocyte differentiation / positive regulation of protein serine/threonine kinase activity / cellular response to calcium ion / protein serine/threonine/tyrosine kinase activity / activation of protein kinase activity / thymus development / neuron differentiation / animal organ morphogenesis / microtubule organizing center / cell cycle arrest / late endosome / scaffold protein binding / chemotaxis / heart development / peptidyl-threonine phosphorylation / positive regulation of peptidyl-serine phosphorylation / protein N-terminus binding / protein tyrosine kinase activity / activation of MAPK activity / protein C-terminus binding / early endosome / positive regulation of ERK1 and ERK2 cascade / negative regulation of gene expression / non-specific serine/threonine protein kinase / MAPK cascade / 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 / 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. / Protein kinase-like domain superfamily / 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. / Protein kinase-like domain superfamily / 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 / Serine/threonine-protein kinase, active site / 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: 4.1 Å
AuthorsPark, E. / Rawson, S. / Li, K. / 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 signaling 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 signaling 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. At present, the structural basis of RAF regulation is poorly understood. Here we describe autoinhibited and active state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer, determined using cryo-electron microscopy (cryo-EM). A 4.1 Å resolution cryo-EM 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 (CRD) occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain (RBD) is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding CRD 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, driving 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
DepositionFeb 11, 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: Dual specificity mitogen-activated protein kinase kinase 1
C: 14-3-3 protein zeta
D: 14-3-3 protein zeta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)193,11610
Polymers191,5544
Non-polymers1,5626
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 3 types, 4 molecules ABCD

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


Mass: 89402.789 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: 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.: 1 / Details: GDC-0623 / 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 , 5 types, 6 molecules

#4: 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.: 1 / Source method: obtained synthetically / Formula: C10H16N5O12P3S
Comment: ATP-gamma-S (energy-carrying molecule analogue) *YM
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Zinc
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Magnesium
#8: 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.: 1 / Source method: obtained synthetically / Formula: C16H14FIN4O3

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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 BRAF/MEK1/14-3-3 with MEK inhibitor
Type: COMPLEX
Details: 14-3-3 is heterodimer of Insect epsilon and zeta. model was generated by Insect zeta sequence as a homo-dimer.
Entity ID: 1, 2, 3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.19 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 0.05 mg/ml / Embedding 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 / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 165298 / Symmetry type: POINT
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.0148978
f_angle_d1.75212119
f_dihedral_angle_d15.8735464
f_chiral_restr0.3261335
f_plane_restr0.0021543

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