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- EMDB-27684: Structure of the PEAK3 pseudokinase homodimer -

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Basic information

Entry
Database: EMDB / ID: EMD-27684
TitleStructure of the PEAK3 pseudokinase homodimer
Map dataReconstruction of the PEAK3 pseudokinase homodimer filtered to 4.9A and sharpened with -105 bFactor.
Sample
  • Complex: PEAK3 pseudokinase homodimer
    • Protein or peptide: Protein PEAK3
Keywordscomplex / pseudokinase / kinase / adapter / SIGNALING PROTEIN
Function / homology: / regulation of cell motility / regulation of actin cytoskeleton organization / regulation of cell shape / actin cytoskeleton / protein kinase activity / focal adhesion / Protein PEAK3
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsTorosyan H / Paul M / Jura N / Verba KA
Funding support United States, 3 items
OrganizationGrant numberCountry
Other governmentQBI Independent Research Fellowship
National Institutes of Health/National Cancer Institute (NIH/NCI)U54 CA209891 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM139636 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3.
Authors: Hayarpi Torosyan / Michael D Paul / Antoine Forget / Megan Lo / Devan Diwanji / Krzysztof Pawłowski / Nevan J Krogan / Natalia Jura / Kliment A Verba /
Abstract: PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK ...PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, which when prevented leads to PEAK3 nuclear enrichment and distinct protein-protein interactions. Altogether, our data demonstrate that PEAK3 dimerization forms an unusual secondary interface for 14-3-3 binding, facilitating 14-3-3 regulation of PEAK3 localization and interactome diversity.
History
DepositionJul 21, 2022-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27684.png

Downloads & links

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Map

FileDownload / File: emd_27684.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the PEAK3 pseudokinase homodimer filtered to 4.9A and sharpened with -105 bFactor.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 320 pix.
= 267.2 Å		0.84 Å/pix.
x 320 pix.
= 267.2 Å		0.84 Å/pix.
x 320 pix.
= 267.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00938
Minimum - Maximum-0.01040972 - 0.035529017
Average (Standard dev.)0.000063914995 (±0.0008550085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 267.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27684_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered, unsharpened half maps

Fileemd_27684_half_map_1.map
Annotationunfiltered, unsharpened half maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered, unsharpened half maps

Fileemd_27684_half_map_2.map
Annotationunfiltered, unsharpened half maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PEAK3 pseudokinase homodimer

EntireName: PEAK3 pseudokinase homodimer
Components
  • Complex: PEAK3 pseudokinase homodimer
    • Protein or peptide: Protein PEAK3

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Supramolecule #1: PEAK3 pseudokinase homodimer

SupramoleculeName: PEAK3 pseudokinase homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 104.58 KDa

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Macromolecule #1: Protein PEAK3

MacromoleculeName: Protein PEAK3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.357031 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSPEPPTEP PEPDNPTWST QPTYSNLGQI RAHLLPSKAC RLRTPGSLST NPEPLPPPLP KKILTRTQSL PTRRTLHPSS IQVQPPRRP FLGSHSVDKS QAAVGPACLP AELTFGPADA PLGLSLRDLH SPEAVHTALA ARQLQGLRTI YARLRARLMG G HPGPCHPG ...String:
MSSPEPPTEP PEPDNPTWST QPTYSNLGQI RAHLLPSKAC RLRTPGSLST NPEPLPPPLP KKILTRTQSL PTRRTLHPSS IQVQPPRRP FLGSHSVDKS QAAVGPACLP AELTFGPADA PLGLSLRDLH SPEAVHTALA ARQLQGLRTI YARLRARLMG G HPGPCHPG HSFRLLDSSP CAESGDALYY RVVRAHEDAW HILVAKVPKP GADVPHPWGL ELQASLSPHF NLQGLCGLVP EG TLPGAPW RGAVALAAEV PERTVAQWLA EACTQPPEEF VWAVALLLLQ LSAALKFLEA WGAALVELRP ENLLLVAPRG CAT TGPPRL LLTDFGRVCL QPPGPPGSPG PHAPQLGSLL RALLSLAAPS TTPLAAGLEL LAAQLTRLRP SASRTRGALQ ALLW GPGPE LRGRGAPLGP WLRALGPWLR VRRGLLVLRL AERAAGGEAP SLEDWLCCEY LAEATESSMG QALALLWDLE GGGGA DYKD DDDKGPV

UniProtKB: Protein PEAK3

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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

Concentration1.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNH2C(CH2OH)3Tris Base
150.0 mMNaClSodium Chloride
2.0 mMC4H10O2S2Dithiothreitol
2.0 mMMgCl2Magnesium Chloride

Details: A final concentration of 0.1% of Octyl-beta-Glucoside (C14H28O6) was added to the sample before freezing.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot time = 7s blot force = 4.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 69.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 32734
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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