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Yorodumi- PDB-7mez: Structure of the phosphoinositide 3-kinase p110 gamma (PIK3CG) p1... -
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-Basic information
Entry | Database: PDB / ID: 7mez | ||||||
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Title | Structure of the phosphoinositide 3-kinase p110 gamma (PIK3CG) p101 (PIK3R5) complex | ||||||
Components |
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Keywords | TRANSFERASE / PI3K / p110 / PIK3CG / PIK3R5 / p101 / phosphoinositide 3-kinase / PIP3 / IMMUNE SYSTEM | ||||||
Function / homology | Function and homology information negative regulation of triglyceride catabolic process / secretory granule localization / natural killer cell chemotaxis / phosphatidylinositol-4-phosphate 3-kinase / neutrophil extravasation / 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 ...negative regulation of triglyceride catabolic process / secretory granule localization / natural killer cell chemotaxis / phosphatidylinositol-4-phosphate 3-kinase / neutrophil extravasation / 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 / 1-phosphatidylinositol-4-phosphate 3-kinase activity / 1-phosphatidylinositol-4,5-bisphosphate 3-kinase activity / dendritic cell chemotaxis / phosphatidylinositol-4,5-bisphosphate 3-kinase / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / mast cell degranulation / 1-phosphatidylinositol-3-kinase activity / Erythropoietin activates Phosphoinositide-3-kinase (PI3K) / hepatocyte apoptotic process / positive regulation of Rac protein signal transduction / phosphatidylinositol-mediated signaling / regulation of cell adhesion mediated by integrin / Synthesis of PIPs at the plasma membrane / phosphatidylinositol phosphate biosynthetic process / regulation of angiogenesis / phosphorylation / T cell proliferation / cellular response to cAMP / GPVI-mediated activation cascade / neutrophil chemotaxis / ephrin receptor binding / phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of endothelial cell migration / G-protein beta/gamma-subunit complex binding / T cell activation / platelet aggregation / positive regulation of cytokine production / positive regulation of MAP kinase activity / endocytosis / G beta:gamma signalling through PI3Kgamma / kinase activity / phospholipase C-activating G protein-coupled receptor signaling pathway / 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 / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / ATP binding / identical protein binding / membrane / nucleus / plasma membrane / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | Homo sapiens (human) Sus scrofa (pig) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å | ||||||
Authors | Burke, J.E. / Dalwadi, U. / Rathinaswamy, M.K. / Yip, C.K. | ||||||
Funding support | Canada, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7mez.cif.gz | 291.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7mez.ent.gz | 226.6 KB | Display | PDB format |
PDBx/mmJSON format | 7mez.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7mez_validation.pdf.gz | 869.4 KB | Display | wwPDB validaton report |
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Full document | 7mez_full_validation.pdf.gz | 886.9 KB | Display | |
Data in XML | 7mez_validation.xml.gz | 50.4 KB | Display | |
Data in CIF | 7mez_validation.cif.gz | 75.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/me/7mez ftp://data.pdbj.org/pub/pdb/validation_reports/me/7mez | HTTPS FTP |
-Related structure data
Related structure data | 23808MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 |
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#2: Protein | 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 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: 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 | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) | |||||||||||||||||||||||||
Buffer solution | pH: 8.5 Details: Freshly prepared gel filtration buffer, filtered through 0.22um filter and degassed | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 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 support | Details: Glow discharged using the Pelco EasiGlow. 15mA Current. Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 1.5s blot time, -5 blot force |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron 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 optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
Software | Name: PHENIX / Version: 1.19.1_4122: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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Image processing | 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 ...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 correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 3762631 Details: Particles were picked using the cryoSPARC template picker | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 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 |
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