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- EMDB-0541: Structure of a MAPK pathway complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0541
TitleStructure of a MAPK pathway complex
Map data
SampleTernary complex of BRAF/MEK1/14-3-3 with MEK inhibitor
  • Serine/threonine-protein kinase B-raf
  • Dual specificity mitogen-activated protein kinase kinase 1
  • 14-3-3 protein zeta
  • (ligand) x 5
Function / homology
Function and homology information


epithelial cell proliferation involved in lung morphogenesis / mitogen-activated protein kinase kinase / labyrinthine layer development / placenta blood vessel development / regulation of axon regeneration / MAP-kinase scaffold activity / cerebellar cortex formation / dUTP catabolic process / regulation of Golgi inheritance / dUMP biosynthetic process ...epithelial cell proliferation involved in lung morphogenesis / mitogen-activated protein kinase kinase / labyrinthine layer development / placenta blood vessel development / regulation of axon regeneration / MAP-kinase scaffold activity / cerebellar cortex formation / dUTP catabolic process / regulation of Golgi inheritance / dUMP biosynthetic process / dUTP diphosphatase / dUTP diphosphatase activity / trachea formation / regulation of early endosome to late endosome transport / face development / thyroid gland development / positive regulation of axonogenesis / regulation of stress-activated MAPK cascade / cellular senescence / Bergmann glial cell differentiation / signal transduction by protein phosphorylation / stress-activated protein kinase signaling cascade / positive regulation of production of miRNAs involved in gene silencing by miRNA / cell motility / MAP kinase kinase activity / ERK1 and ERK2 cascade / regulation of mitotic cell cycle / keratinocyte differentiation / protein serine/threonine kinase activator activity / microtubule organizing center / activation of protein kinase activity / thymus development / positive regulation of protein serine/threonine kinase activity / protein serine/threonine/tyrosine kinase activity / neuron differentiation / cell cycle arrest / chemotaxis / scaffold protein binding / late endosome / peptidyl-threonine phosphorylation / protein N-terminus binding / heart development / protein tyrosine kinase activity / protein C-terminus binding / activation of MAPK activity / early endosome / positive regulation of ERK1 and ERK2 cascade / MAPK cascade / negative regulation of gene expression / protein kinase activity / negative regulation of cell population proliferation / focal adhesion / protein serine/threonine kinase activity / positive regulation of gene expression / protein phosphorylation / signal transduction / Golgi apparatus / positive regulation of transcription, DNA-templated / endoplasmic reticulum / magnesium ion binding / mitochondrion / ATP binding / plasma membrane / nucleus / cytosol / cytoplasm
Deoxyuridine triphosphate nucleotidohydrolase / Protein kinase domain / 14-3-3 domain superfamily / dUTPase-like superfamily / dUTPase, trimeric / dUTPase-like / 14-3-3 domain / 14-3-3 protein, conserved site / Protein kinase, ATP binding site / Protein kinase-like domain superfamily ...Deoxyuridine triphosphate nucleotidohydrolase / Protein kinase domain / 14-3-3 domain superfamily / dUTPase-like superfamily / dUTPase, trimeric / dUTPase-like / 14-3-3 domain / 14-3-3 protein, conserved site / Protein kinase, ATP binding site / Protein kinase-like domain superfamily / 14-3-3 protein / Serine/threonine-protein kinase, active site
Deoxyuridine 5'-triphosphate nucleotidohydrolase / Dual specificity mitogen-activated protein kinase kinase 1 / 14-3-3 protein zeta
Biological speciesHomo sapiens (human) / Beet armyworm (beet armyworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsPark E / Rawson S / Li K / Jeon H / Eck MJ
Funding support United States, 2 items
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 ReportPDB-ID: 6nyb

SummaryFull reportAbout validation report
History
DepositionFeb 11, 2019-
Header (metadata) releaseMar 27, 2019-
Map releaseOct 9, 2019-
UpdateApr 22, 2020-
Current statusApr 22, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.048
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nyb
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0541.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 212. Å
1.06 Å/pix.
x 200 pix.
= 212. Å
1.06 Å/pix.
x 200 pix.
= 212. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.048 / Movie #1: 0.048
Minimum - Maximum-0.15667647 - 0.21985821
Average (Standard dev.)0.00056338793 (±0.0067066685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.000212.000212.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1570.2200.001

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Supplemental data

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Sample components

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Entire Ternary complex of BRAF/MEK1/14-3-3 with MEK inhibitor

EntireName: Ternary complex of BRAF/MEK1/14-3-3 with MEK inhibitor
Details: 14-3-3 is heterodimer of Insect epsilon and zeta. model was generated by Insect zeta sequence as a homo-dimer.
Number of components: 9

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Component #1: protein, Ternary complex of BRAF/MEK1/14-3-3 with MEK inhibitor

ProteinName: Ternary complex of BRAF/MEK1/14-3-3 with MEK inhibitor
Details: 14-3-3 is heterodimer of Insect epsilon and zeta. model was generated by Insect zeta sequence as a homo-dimer.
Recombinant expression: No
MassExperimental: 190 kDa
SourceSpecies: Homo sapiens (human)

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Component #2: protein, Serine/threonine-protein kinase B-raf

ProteinName: Serine/threonine-protein kinase B-raf / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 89.402789 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, Dual specificity mitogen-activated protein kinase kinase 1

ProteinName: Dual specificity mitogen-activated protein kinase kinase 1
Details: GDC-0623 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 45.934543 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #4: protein, 14-3-3 protein zeta

ProteinName: 14-3-3 protein zeta / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 28.108514 kDa
SourceSpecies: Beet armyworm (beet armyworm)

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Component #5: ligand, PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

LigandName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.523247 kDa

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Component #6: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #7: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Component #8: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #9: ligand, 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imida...

LigandName: 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.45621 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.05 mg/mL / pH: 7.4
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: OTHER
LensCs: 2.7 mm / Imaging mode: OTHER
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 165298
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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