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- EMDB-23812: Structure of the apo phosphoinositide 3-kinase p110 gamma (PIK3CG... -

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Basic information

Entry
Database: EMDB / ID: EMD-23812
TitleStructure of the apo phosphoinositide 3-kinase p110 gamma (PIK3CG) p101 (PIK3R5) complex
Map data
Sample
  • Complex: Complex of p110 gamma with p101
Function / homology
Function and homology information


: / secretory granule localization / negative regulation of triglyceride catabolic process / natural killer cell chemotaxis / neutrophil extravasation / phosphatidylinositol-4-phosphate 3-kinase / regulation of calcium ion transmembrane transport / positive regulation of acute inflammatory response / respiratory burst involved in defense response / negative regulation of cardiac muscle contraction ...: / secretory granule localization / negative regulation of triglyceride catabolic process / natural killer cell chemotaxis / neutrophil extravasation / phosphatidylinositol-4-phosphate 3-kinase / regulation of calcium ion transmembrane transport / positive regulation of acute inflammatory response / respiratory burst involved in defense response / negative regulation of cardiac muscle contraction / 1-phosphatidylinositol-3-kinase regulator activity / T cell chemotaxis / negative regulation of fibroblast apoptotic process / phosphatidylinositol 3-kinase complex, class IB / sphingosine-1-phosphate receptor signaling pathway / phosphatidylinositol 3-kinase complex / dendritic cell chemotaxis / 1-phosphatidylinositol-4-phosphate 3-kinase activity / 1-phosphatidylinositol-4,5-bisphosphate 3-kinase activity / phosphatidylinositol-4,5-bisphosphate 3-kinase / phosphatidylinositol 3-kinase / phosphatidylinositol 3-kinase complex, class IA / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / mast cell degranulation / Erythropoietin activates Phosphoinositide-3-kinase (PI3K) / hepatocyte apoptotic process / positive regulation of Rac protein signal transduction / regulation of cell adhesion mediated by integrin / Synthesis of PIPs at the plasma membrane / phosphatidylinositol phosphate biosynthetic process / centriolar satellite / regulation of angiogenesis / T cell proliferation / cellular response to cAMP / GPVI-mediated activation cascade / T cell activation / ephrin receptor binding / neutrophil chemotaxis / positive regulation of endothelial cell migration / phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of cytokine production / G-protein beta/gamma-subunit complex binding / positive regulation of MAP kinase activity / platelet aggregation / endocytosis / G beta:gamma signalling through PI3Kgamma / Signaling by CSF1 (M-CSF) in myeloid cells / kinase activity / positive regulation of cytosolic calcium ion concentration / angiogenesis / adaptive immune response / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / non-specific serine/threonine protein kinase / protein kinase activity / immune response / inflammatory response / G protein-coupled receptor signaling pathway / phosphorylation / protein serine kinase activity / innate immune response / protein serine/threonine kinase activity / ATP binding / membrane / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Phosphoinositide 3-kinase regulatory subunit 5/6 / Phosphoinositide 3-kinase gamma adapter protein p101 subunit / PIK3 catalytic subunit gamma, adaptor-binding domain / PIK3 catalytic subunit gamma adaptor-binding domain / Phosphatidylinositol 3-kinase, adaptor-binding domain / Phosphatidylinositol 3-kinase adaptor-binding (PI3K ABD) domain profile. / 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. ...Phosphoinositide 3-kinase regulatory subunit 5/6 / Phosphoinositide 3-kinase gamma adapter protein p101 subunit / PIK3 catalytic subunit gamma, adaptor-binding domain / PIK3 catalytic subunit gamma adaptor-binding domain / Phosphatidylinositol 3-kinase, adaptor-binding domain / Phosphatidylinositol 3-kinase adaptor-binding (PI3K ABD) domain profile. / 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. / Phosphoinositide 3-kinase C2 / Phosphoinositide 3-kinase, region postulated to contain C2 domain / C2 phosphatidylinositol 3-kinase-type domain / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical 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 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform / Phosphoinositide 3-kinase regulatory subunit 5
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsBurke JE / Dalwadi U / Rathinaswamy MK / Yip CK
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)168998 Canada
CitationJournal: Sci Adv / Year: 2021
Title: Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation.
Authors: Manoj K Rathinaswamy / Udit Dalwadi / Kaelin D Fleming / Carson Adams / Jordan T B Stariha / Els Pardon / Minkyung Baek / Oscar Vadas / Frank DiMaio / Jan Steyaert / Scott D Hansen / Calvin ...Authors: Manoj K Rathinaswamy / Udit Dalwadi / Kaelin D Fleming / Carson Adams / Jordan T B Stariha / Els Pardon / Minkyung Baek / Oscar Vadas / Frank DiMaio / Jan Steyaert / Scott D Hansen / Calvin K Yip / John E Burke /
Abstract: The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function ...The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.
History
DepositionApr 8, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.892
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.892
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23812.png

Downloads & links

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Map

FileDownload / File: emd_23812.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.079 Å
Density
Contour LevelBy AUTHOR: 0.892 / Movie #1: 0.892
Minimum - Maximum-3.0035362 - 5.170192
Average (Standard dev.)0.00012388699 (±0.098279685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 323.7 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0791.0791.079
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z323.700323.700323.700
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-3.0045.1700.000

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Supplemental data

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Sample components

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Entire : Complex of p110 gamma with p101

EntireName: Complex of p110 gamma with p101
Components
  • Complex: Complex of p110 gamma with p101

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Supramolecule #1: Complex of p110 gamma with p101

SupramoleculeName: Complex of p110 gamma with p101 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: p110 subunit is from homo sapiens, p101 is from Sus scrofa
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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

Concentration0.4 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
20.0 mMTrisHClTris(hydroxymethyl)aminomethane-Hydrochloric acid
100.0 mMNaClSodium chlorideSodium Chloride
50.0 mM(NH4)2SO4Ammonium Sulfate
0.5 mMTCEPTris(2-carboxyethyl)phosphine

Details: Freshly prepared gel filtration buffer, filtered through 0.22um filter and degassed
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
Details: Glow discharged using a Pelco EasiGlow. 15mA current.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 1.5s blot time, -5 blot force.
DetailsSpecimen was a 1:1 molar ratio of p110g-p101, purified to homogeneity by gel filtration.

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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 / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPNCC Krios 4
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6153 / Average electron dose: 50.0 e/Å2
Details: Movies were collected in super-resolution mode set to collect 3 shots per grid hole over 5 holes by beam-shift before applying a stage shift.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4792176
Details: Particles were picked using the cryoSPARC template picker
CTF correctionSoftware - Name: cryoSPARC (ver. 2.18)
Startup modelType of model: NONE / Details: Ab initio reconstruction with 2 classes
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.18) / Details: cryoSPARC SGD-based ab intio reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.18) / Details: cryoSPARC non-uniform refinement
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.18)
Details: Final reconstruction was generated in cryoSPARC v2.18 using the (now) legacy NU-refinement job.
Number images used: 196390
DetailsAll data processing carried out using cryoSPARC v2.18 or newer. Movies were subjected to full-frame motion correction and 2x2 binning. CTFs of the resulting micrographs were estimated using patch CTF estimation. Template picking using 2D averages low-pass filtered to 20A was carried out before 2 rounds of 2D classification. Best particles were refined by local motion correction, then subjected to 2 more rounds of 2D classification. Best particles were then classified further by 2 rounds of 3D classification. The best class/particles were further refined by homogeneous refinement, followed by a final non-uniform refinement step.
FSC plot (resolution estimation)

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