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- PDB-7mez: Structure of the phosphoinositide 3-kinase p110 gamma (PIK3CG) p1... -

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Basic information

Entry
Database: PDB / ID: 7mez
TitleStructure of the phosphoinositide 3-kinase p110 gamma (PIK3CG) p101 (PIK3R5) complex
Components
  • Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform
  • Phosphoinositide 3-kinase regulatory subunit 5
KeywordsTRANSFERASE / PI3K / p110 / PIK3CG / PIK3R5 / p101 / phosphoinositide 3-kinase / PIP3 / IMMUNE SYSTEM
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 / positive regulation of acute inflammatory response / respiratory burst involved in defense response / negative regulation of cardiac muscle contraction / regulation of calcium ion transmembrane transport ...: / secretory granule localization / negative regulation of triglyceride catabolic process / natural killer cell chemotaxis / neutrophil extravasation / phosphatidylinositol-4-phosphate 3-kinase / positive regulation of acute inflammatory response / respiratory burst involved in defense response / negative regulation of cardiac muscle contraction / regulation of calcium ion transmembrane transport / 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, class IA / 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-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-mediated signaling / phosphatidylinositol phosphate biosynthetic process / regulation of angiogenesis / T cell proliferation / cellular response to cAMP / GPVI-mediated activation cascade / T cell activation / ephrin receptor binding / positive regulation of endothelial cell migration / phosphatidylinositol 3-kinase/protein kinase B signal transduction / neutrophil chemotaxis / positive regulation of cytokine production / positive regulation of MAP kinase activity / G-protein beta/gamma-subunit complex binding / 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 / inflammatory response / immune response / G protein-coupled receptor signaling pathway / phosphorylation / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / ATP binding / identical protein binding / membrane / nucleus / plasma membrane / cytoplasm / cytosol
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 catalytic subunit gamma isoform
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsBurke, J.E. / Dalwadi, U. / Rathinaswamy, M.K. / Yip, C.K.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)168998 Canada
CitationJournal: Structure / Year: 2021
Title: HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.
Authors: Manoj K Rathinaswamy / Kaelin D Fleming / Udit Dalwadi / Els Pardon / Noah J Harris / Calvin K Yip / Jan Steyaert / John E Burke /
Abstract: There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) ...There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.
History
DepositionApr 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 14, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.journal_volume / _citation.pdbx_database_id_DOI ..._citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform
B: Phosphoinositide 3-kinase regulatory subunit 5


Theoretical massNumber of molelcules
Total (without water)224,0992
Polymers224,0992
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform / PI3-kinase subunit gamma / PI3K-gamma / PI3Kgamma / PtdIns-3-kinase subunit gamma / ...PI3-kinase subunit gamma / PI3K-gamma / PI3Kgamma / PtdIns-3-kinase subunit gamma / Phosphatidylinositol 4 / 5-bisphosphate 3-kinase 110 kDa catalytic subunit gamma / PtdIns-3-kinase subunit p110-gamma / p110gamma / Phosphoinositide-3-kinase catalytic gamma polypeptide / Serine/threonine protein kinase PIK3CG / p120-PI3K


Mass: 126627.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3CG / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P48736, phosphatidylinositol 3-kinase, phosphatidylinositol-4,5-bisphosphate 3-kinase, phosphatidylinositol-4-phosphate 3-kinase, non-specific serine/threonine protein kinase
#2: Protein Phosphoinositide 3-kinase regulatory subunit 5 / PI3-kinase regulatory subunit 5 / IB PI3-kinase p101 subunit / PI3-kinase p101 subunit / ...PI3-kinase regulatory subunit 5 / IB PI3-kinase p101 subunit / PI3-kinase p101 subunit / Phosphatidylinositol 4 / 5-bisphosphate 3-kinase regulatory subunit / PtdIns-3-kinase regulatory subunit / PtdIns-3-kinase p101 / p101-PI3K


Mass: 97471.805 Da / Num. of mol.: 1 / Mutation: G473R, V530A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig) / Gene: PIK3R5 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O02696

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of p110 gamma with p101 and a p101 binding nanobody
Type: COMPLEX
Details: Nanobody binds to GBD domain, but was too fleixble to be built in the atomic model
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Sus scrofa (pig)9823
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8.5
Details: Freshly prepared gel filtration buffer, filtered through 0.22um filter and degassed
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(hydroxymethyl)aminomethane-Hydrochloric acidTrisHCl1
2100 mMSodium ChlorideNaCl1
350 mMAmmonium Sulfate(NH4)2SO41
40.5 mMTris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 0.45 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Specimen was a 1:1:1 molar ratio of p110g-p101-nanobody, purified to homogeneity by gel filtration.
Specimen supportDetails: Glow discharged using the Pelco EasiGlow. 15mA Current.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 1.5s blot time, -5 blot force

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 36.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6808
Details: Movies were collected in super-resolution mode set to collect 3 shots per grid hole over 9 holes by beam-shift before applying a stage shift.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3CTF correction
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
13cryoSPARC33D reconstruction
Image processingDetails: All data processing carried out using cryoSPARC v2.18 or newer. Movies were subjected to patch motion correction and 2x2 binning. CTFs of the resulting micrographs were estimated using patch ...Details: All data processing carried out using cryoSPARC v2.18 or newer. Movies were subjected to patch 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 1 round of 2D and 1 round of 3D classification. Best particles were refined by local motion correction, then subjected to 1 more round of 3D classification. Best class/particles were refined by homogeneous refinement, followed by CTF/defocus refinement and a final non-uniform refinement step.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3762631
Details: Particles were picked using the cryoSPARC template picker
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 320179 / Details: cryoSPARC local refinement job (L) from v3.0 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312647
ELECTRON MICROSCOPYf_angle_d0.5517142
ELECTRON MICROSCOPYf_dihedral_angle_d4.6821660
ELECTRON MICROSCOPYf_chiral_restr0.0421933
ELECTRON MICROSCOPYf_plane_restr0.0042185

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