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- EMDB-40253: Truncated Braf/Mek/14-3-3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-40253
TitleTruncated Braf/Mek/14-3-3 complex
Map data(del1-155)Braf/Mek/14-3-3 complex
Sample
  • Complex: Truncated Braf/Mek/14-3-3 complex
Keywordskinase complex / TRANSFERASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsEck MJ / Jeon H / Park E / Rawson S
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P50CA165962 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R35CA242461 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R50CA220830 United States
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM structure of a RAS/RAF recruitment complex.
Authors: Eunyoung Park / Shaun Rawson / Anna Schmoker / Byeong-Won Kim / Sehee Oh / Kangkang Song / Hyesung Jeon / Michael J Eck /
Abstract: RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF ...RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.
History
DepositionMar 30, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40253.png

Downloads & links

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Map

FileDownload / File: emd_40253.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation(del1-155)Braf/Mek/14-3-3 complex
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.321
Minimum - Maximum-0.6638111 - 1.3207767
Average (Standard dev.)0.001511512 (±0.05001029)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_40253_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: (del1-155)Braf/Mek/14-3-3 complex

Fileemd_40253_additional_1.map
Annotation(del1-155)Braf/Mek/14-3-3 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: (del1-155)Braf/Mek/14-3-3 complex

Fileemd_40253_half_map_1.map
Annotation(del1-155)Braf/Mek/14-3-3 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: (del1-155)Braf/Mek/14-3-3 complex

Fileemd_40253_half_map_2.map
Annotation(del1-155)Braf/Mek/14-3-3 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Truncated Braf/Mek/14-3-3 complex

EntireName: Truncated Braf/Mek/14-3-3 complex
Components
  • Complex: Truncated Braf/Mek/14-3-3 complex

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Supramolecule #1: Truncated Braf/Mek/14-3-3 complex

SupramoleculeName: Truncated Braf/Mek/14-3-3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: (del1-155)Braf/Mek/14-3-3 complex
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 185 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4 / Component - Concentration: 150.0 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride
Details: 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP, 50 uM ATP-gammaS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Detailsmonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 130000
Specialist opticsPhase plate: VOLTA PHASE PLATE
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3252 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 4.0.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 91490
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER

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