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- EMDB-12191: Cryo-EM structure of the Phosphatidylinositol 3-kinase type 2a (P... -

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Basic information

Entry
Database: EMDB / ID: EMD-12191
TitleCryo-EM structure of the Phosphatidylinositol 3-kinase type 2a (PI3KC2a) of the class II PI3K family
Map datacryosparc refined map
Sample
  • Complex: Class II PI3KA
Function / homology
Function and homology information


negative regulation of zinc ion transmembrane transport / Synthesis of PIPs at the early endosome membrane / Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the Golgi membrane / Synthesis of PIPs at the plasma membrane / autophagosome organization / Golgi Associated Vesicle Biogenesis / phosphatidylinositol-4-phosphate 3-kinase / Clathrin-mediated endocytosis / phosphatidylinositol 3-kinase complex ...negative regulation of zinc ion transmembrane transport / Synthesis of PIPs at the early endosome membrane / Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the Golgi membrane / Synthesis of PIPs at the plasma membrane / autophagosome organization / Golgi Associated Vesicle Biogenesis / phosphatidylinositol-4-phosphate 3-kinase / Clathrin-mediated endocytosis / phosphatidylinositol 3-kinase complex / 1-phosphatidylinositol-4-phosphate 3-kinase activity / 1-phosphatidylinositol-4,5-bisphosphate 3-kinase activity / phosphatidylinositol-4,5-bisphosphate 3-kinase / clathrin-coated vesicle / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / clathrin binding / exocytosis / phosphatidylinositol-mediated signaling / phosphatidylinositol 3-kinase/protein kinase B signal transduction / cellular response to starvation / phosphatidylinositol binding / macroautophagy / trans-Golgi network / endocytosis / vesicle / ATP binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
PI3K-C2-alpha, catalytic domain / Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha, PX domain / PhoX homologous domain, present in p47phox and p40phox. / PX domain profile. / PX domain / Phox homology / PX domain superfamily / PI3-kinase family, Ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain / PI3-kinase family, ras-binding domain ...PI3K-C2-alpha, catalytic domain / Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha, PX domain / PhoX homologous domain, present in p47phox and p40phox. / PX domain profile. / PX domain / Phox homology / PX domain superfamily / PI3-kinase family, Ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain / PI3-kinase family, ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain profile. / C2 phosphatidylinositol 3-kinase-type domain / Phosphoinositide 3-kinase C2 / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, region postulated to contain C2 domain / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / C2 domain superfamily / Armadillo-type fold / Ubiquitin-like domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsLo WT / Zhang Y / Vadas O / Belabed H / Roeske Y / Gulluni F / De Santis MC / Vujicic Zagar A / Stephanowitz H / Hirsch E ...Lo WT / Zhang Y / Vadas O / Belabed H / Roeske Y / Gulluni F / De Santis MC / Vujicic Zagar A / Stephanowitz H / Hirsch E / Liu F / Daumke O / Nazare M / Kudryashev M / Haucke V
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structural basis of phosphatidylinositol 3-kinase C2α function.
Authors: Wen-Ting Lo / Yingyi Zhang / Oscar Vadas / Yvette Roske / Federico Gulluni / Maria Chiara De Santis / Andreja Vujicic Zagar / Heike Stephanowitz / Emilio Hirsch / Fan Liu / Oliver Daumke / ...Authors: Wen-Ting Lo / Yingyi Zhang / Oscar Vadas / Yvette Roske / Federico Gulluni / Maria Chiara De Santis / Andreja Vujicic Zagar / Heike Stephanowitz / Emilio Hirsch / Fan Liu / Oliver Daumke / Misha Kudryashev / Volker Haucke /
Abstract: Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, ...Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, viral replication, platelet formation and a role in mitosis. The molecular basis of these activities of PI3KC2α is poorly understood. Here, we report high-resolution crystal structures as well as a 4.4-Å cryogenic-electron microscopic (cryo-EM) structure of PI3KC2α in active and inactive conformations. We unravel a coincident mechanism of lipid-induced activation of PI3KC2α at membranes that involves large-scale repositioning of its Ras-binding and lipid-binding distal Phox-homology and C-C2 domains, and can serve as a model for the entire class II PI3K family. Moreover, we describe a PI3KC2α-specific helical bundle domain that underlies its scaffolding function at the mitotic spindle. Our results advance our understanding of PI3K biology and pave the way for the development of specific inhibitors of class II PI3K function with wide applications in biomedicine.
History
DepositionJan 12, 2021-
Header (metadata) releaseMar 9, 2022-
Map releaseMar 9, 2022-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12191.png

Downloads & links

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Map

FileDownload / File: emd_12191.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryosparc refined map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 260 pix.
= 217.62 Å		0.84 Å/pix.
x 260 pix.
= 217.62 Å		0.84 Å/pix.
x 260 pix.
= 217.62 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.837 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.09764164 - 0.23457181
Average (Standard dev.)0.00014710799 (±0.009579717)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 217.62001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8370.8370.837
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z217.620217.620217.620
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0980.2350.000

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Supplemental data

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Mask #1

Fileemd_12191_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: RELION postprocess map

Fileemd_12191_additional_1.map
AnnotationRELION postprocess map
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AxesZYX

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Histogram

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Half map: half map 1

Fileemd_12191_half_map_1.map
Annotationhalf map 1
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_12191_half_map_2.map
Annotationhalf map 2
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Sample components

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Entire : Class II PI3KA

EntireName: Class II PI3KA
Components
  • Complex: Class II PI3KA

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Supramolecule #1: Class II PI3KA

SupramoleculeName: Class II PI3KA / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightExperimental: 150 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

Concentration0.8 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris(hydroxymethyl)aminomethane hydrochloride
100.0 mMNaClSodium chloride

Details: The buffer containing 20 mM Tris-HCl, 100 mM NaCl at pH 7.4.
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
Details: QUANTIFOIL Holey Au-carbon-R2/2 specimen grids were used.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: The sample was vitrified by plunge-freezing into liquid ethane using a Mark IV Vitrobot device (Thermo Fisher Scientific), blotting force 20, blotting time 4.5-5.5s..
DetailsThe purified sample was diluted to 0.8 mg/ml into buffer containing 20 mM Tris-HCl, 100 mM NaCl at pH 7.4.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 79.8 K / Max: 89.9 K
Specialist opticsSpherical aberration corrector: no / Chromatic aberration corrector: no / Energy filter - Slit width: 20 eV
DetailsGrids were screened first to check ice thickness and particle distribution.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number real images: 10433 / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2
Details: Images were collected with 50 frames over 60 electrons per squared angstrom.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.3000000000000003 µm / Calibrated defocus min: 1.2 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.5 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

DetailsThe images were collected and processed with cryosparc.
Particle selectionNumber selected: 2752000 / Details: Particles were auto picked with blob picker.
CTF correctionSoftware - Name: cryoSPARC (ver. V2.12) / Software - details: Patch CTF estimation
Details: CTF estimation was performed after motion correction. And Ctf local refine was performed after 3D reconstruction, in cryosparc.
Startup modelType of model: NONE
Details: Ab-initio reconstruction from 2D selected particles
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. V2.12) / Software - details: Homogenous refinement / Number images used: 601000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. V2.12)
Details: stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms
Final 3D classificationNumber classes: 100 / Avg.num./class: 8490 / Software - Name: cryoSPARC (ver. v2.12) / Software - details: 2D classification
FSC plot (resolution estimation)

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