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- EMDB-40652: Phosphoinositide phosphate 3 kinase gamma bound with ADP -

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Basic information

Entry
Database: EMDB / ID: EMD-40652
TitlePhosphoinositide phosphate 3 kinase gamma bound with ADP
Map data
Sample
  • Complex: PI3K-gamma-ADPBeF3
    • Protein or peptide: phosphatidylinositol-4,5-bisphosphate 3-kinase
    • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 5
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsPhosphoinositide 3-Kinase / Chemotaxis / Cancer / SIGNALING PROTEIN
Function / homology
Function and homology information


1-phosphatidylinositol-3-kinase regulator activity / phosphatidylinositol 3-kinase complex, class IB / phosphatidylinositol-mediated signaling / phosphatidylinositol-4,5-bisphosphate 3-kinase / phosphatidylinositol phosphate biosynthetic process / kinase activity / phosphorylation / nucleus / plasma membrane / 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
Phosphoinositide 3-kinase regulatory subunit 5 / phosphatidylinositol-4,5-bisphosphate 3-kinase
Similarity search - Component
Biological speciesSus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsChen C-L / Tesmer JJG / Bandekar SJ / Cash J
Funding support United States, 1 items
OrganizationGrant numberCountry
American Heart Association834497 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Molecular basis for Gβγ-mediated activation of phosphoinositide 3-kinase γ.
Authors: Chun-Liang Chen / Ramizah Syahirah / Sandeep K Ravala / Yu-Chen Yen / Thomas Klose / Qing Deng / John J G Tesmer /
Abstract: The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. ...The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. PI3Kγ is activated by Gβγ heterodimers released from G protein-coupled receptors responding to extracellular signals. Here we determined cryo-electron microscopy structures of Sus scrofa PI3Kγ-human Gβγ complexes in the presence of substrates/analogs, revealing two Gβγ binding sites: one on the p110γ helical domain and another on the p101 C-terminal domain. Comparison with PI3Kγ alone reveals conformational changes in the kinase domain upon Gβγ binding that are similar to Ras·GTP-induced changes. Assays of variants perturbing the Gβγ binding sites and interdomain contacts altered by Gβγ binding suggest that Gβγ recruits the enzyme to membranes and allosterically regulates activity via both sites. Studies of zebrafish neutrophil migration align with these findings, paving the way for in-depth investigation of Gβγ-mediated activation mechanisms in this enzyme family and drug development for PI3Kγ.
History
DepositionApr 28, 2023-
Header (metadata) releaseApr 3, 2024-
Map releaseApr 3, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40652.png

Downloads & links

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Map

FileDownload / File: emd_40652.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.47695974 - 0.98189706
Average (Standard dev.)0.00021193727 (±0.022096759)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40652_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40652_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PI3K-gamma-ADPBeF3

EntireName: PI3K-gamma-ADPBeF3
Components
  • Complex: PI3K-gamma-ADPBeF3
    • Protein or peptide: phosphatidylinositol-4,5-bisphosphate 3-kinase
    • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 5
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: PI3K-gamma-ADPBeF3

SupramoleculeName: PI3K-gamma-ADPBeF3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 210 kDa/nm

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Macromolecule #1: phosphatidylinositol-4,5-bisphosphate 3-kinase

MacromoleculeName: phosphatidylinositol-4,5-bisphosphate 3-kinase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 127.573531 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHELE NYEQPVVLRE DNRRRRRRMK PRSTAASLSS MELIPIEFVL PTSQRNTKTP ETALLHVAGH GNVEQMKAQV WLRALETSV SADFYHRLGP DHFLLLYQKK GQWYEIYDKY QVVQTLDCLR YWKVLHRSPG QIHVVQRHAP SEETLAFQRQ L NALIGYDV ...String:
MHHHHHHELE NYEQPVVLRE DNRRRRRRMK PRSTAASLSS MELIPIEFVL PTSQRNTKTP ETALLHVAGH GNVEQMKAQV WLRALETSV SADFYHRLGP DHFLLLYQKK GQWYEIYDKY QVVQTLDCLR YWKVLHRSPG QIHVVQRHAP SEETLAFQRQ L NALIGYDV TDVSNVHDDE LEFTRRRLVT PRMAEVAGRD PKLYAMHPWV TSKPLPEYLL KKITNNCVFI VIHRSTTSQT IK VSADDTP GTILQSFFTK MAKKKSLMDI PESQNERDFV LRVCGRDEYL VGETPIKNFQ WVRQCLKNGE EIHLVLDTPP DPA LDEVRK EEWPLVDDCT GVTGYHEQLT IHGKDHESVF TVSLWDCDRK FRVKIRGIDI PVLPRTADLT VFVEANIQYG QQVL CQRRT SPKPFTEEVL WNVWLEFSIK IKDLPKGALL NLQIYCGKAP ALSGKTSAEM PSPESKGKAQ LLYYVNLLLI DHRFL LRHG EYVLHMWQLS GKGEDQGSFN ADKLTSATNP DKENSMSISI LLDNYCHPIA LPKHRPTPDP EGDRVRAEMP NQLRKQ LEA IIATDPLNPL TAEDKELLWH FRYESLKDPK AYPKLFSSVK WGQQEIVAKT YQLLAKREVW DQSALDVGLT MQLLDCN FS DENVRAIAVQ KLESLEDDDV LHYLLQLVQA VKFEPYHDSA LARFLLKRGL RNKRIGHFLF WFLRSEIAQS RHYQQRFA V ILEAYLRGCG TAMLHDFTQQ VQVIDMLQKV TIDIKSLSAE KYDVSSQVIS QLKQKLENLQ NLNLPQSFRV PYDPGLKAG ALVIEKCKVM ASKKKPLWLE FKCADPTALS NETIGIIFKH GDDLRQDMLI LQILRIMESI WETESLDLCL LPYGCISTGD KIGMIEIVK DATTIAKIQQ STVGNTGAFK DEVLSHWLKE KCPIEEKFQA AVERFVYSCA GYCVATFVLG IGDRHNDNIM I SETGNLFH IDFGHILGNY KSFLGINKER VPFVLTPDFL FVMGTSGKKT SLHFQKFQDV CVKAYLALRH HTNLLIILFS MM LMTGMPQ LTSKEDIEYI RDALTVGKSE EDAKKYFLDQ IEVCRDKGWT VQFNWFLHLV LGIKQGEKHS A

UniProtKB: phosphatidylinositol-4,5-bisphosphate 3-kinase

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Macromolecule #2: Phosphoinositide 3-kinase regulatory subunit 5

MacromoleculeName: Phosphoinositide 3-kinase regulatory subunit 5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 98.497773 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MQPGATTCTE DRIQHALERC LHGLSLSRRS TSWSAGLCLN CWSLQELVSR DPGHFLILLE QILQKTREVQ EKGTYDLLAP LALLFYSTV LCTPHFPPDS DLLLKAARTY HRFLTWPVPY CSICQELLTF IDAELKAPGI SYQRLVRAEQ GLSTRSHRSS T VTVLLLNP ...String:
MQPGATTCTE DRIQHALERC LHGLSLSRRS TSWSAGLCLN CWSLQELVSR DPGHFLILLE QILQKTREVQ EKGTYDLLAP LALLFYSTV LCTPHFPPDS DLLLKAARTY HRFLTWPVPY CSICQELLTF IDAELKAPGI SYQRLVRAEQ GLSTRSHRSS T VTVLLLNP VEVQAEFLDV ADKLSTPGPS PHSAYITLLL HAFQATFGAH CDLSGLHRRL QSKTLAELEA IFTETAEAQE LA SGIGDAA EARQWLRTKL QAVGEKAGFP GVLDTAKPGK LRTIPIPVAR CYTYSWNQDS FDILQEILLK EQELLQPEIL DDE EDEDEE DEEEDLDADG HCAERDSVLS TGSAASHAST LSLASSQASG PTLSRQLLTS FVSGLSDGVD SGYMEDIEES AYER PRRPG GHERRGHRRP GQKFNRIYKL FKSTSQMVLR RDSRSLEGSP DSGPPLRRAG SLCSPLDSPT LPPSRAQRSR SLPQP KLSP QLPGWLLAPA SRHQRRRPFL SGDEDPKAST LRVVVFGSDR ISGKVARAYS NLRRLENNRP LLTRFFKLQF FYVPVK RSR GTGTPTSPAP RSQTPPLPTD APRHPGPAEL GAAPWEESTN DISHYLGMLD PWYERNVLGL MHLPPEVLCQ SLKAEPR PL EGSPAQLPIL ADMLLYYCRF AARPVLLQVY QTELTFITGE KTTEIFIHSL ELGHSAATRA IKASGPGSKR LGIDGDRE A VPLTLQIIYS KGAISGRSRW SNMEKLCTSV NLSKACRQQE ELDSSTEALT LNLTEVVKRQ TPKSKKGFNQ ISTSQIKVD KVQIIGSNSC PFAVCLDQDE RKILQSVIRC EVSPCYKPEK SSLCPPPQRP SYPPAPATPD LCSLLCLPIM TFSGALPGGG GSDYKDDDD K

UniProtKB: Phosphoinositide 3-kinase regulatory subunit 5

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
200.0 mMNaClSodium chlorideSodium Chloride
10.0 mMMgCl2Magnesium Chloride
1.0 mMC10H16N5O10P2ADPAdenosine diphosphate
5.0 mMBeF3Beryllium Fluoride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 2.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum ER / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Alignment procedureComa free - Residual tilt: 0.01 mrad
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Average exposure time: 3.12 sec. / Average electron dose: 55.0 e/Å2
Details: Images were collected in movie-mode at 40 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab-initio model generated from cryoSPARC
Initial angle assignmentType: COMMON LINE / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: COMMON LINE / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 92933
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1e8x

chain_id: A, source_name: SwissModel, initial_model_type: in silico model
chain_id: B, source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: correlation coefficient
Output model

PDB-8sob:
Phosphoinositide phosphate 3 kinase gamma bound with ADP

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