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Yorodumi- EMDB-2846: Three-dimensional structure of the autophagic phosphatidylinosito... -
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-Basic information
Entry | Database: EMDB / ID: EMD-2846 | |||||||||
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Title | Three-dimensional structure of the autophagic phosphatidylinositol 3-kinase complex. | |||||||||
Map data | Three-dimensional structure of the autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1. | |||||||||
Sample |
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Keywords | kinase / lipid kinase / protein kinase / autophagy / phosphatidylinositol 3-kinase / PI3K / phosphatidylinositol 3-phosphate / PI(3)P | |||||||||
Function / homology | Function and homology information extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / regulation of triglyceride metabolic process / extrinsic component of phagophore assembly site membrane / nucleus-vacuole junction / cellular response to aluminum ion / Toll Like Receptor 9 (TLR9) Cascade / protein lipidation / Synthesis of PIPs at the late endosome membrane / phosphatidylinositol 3-kinase complex, class III ...extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / regulation of triglyceride metabolic process / extrinsic component of phagophore assembly site membrane / nucleus-vacuole junction / cellular response to aluminum ion / Toll Like Receptor 9 (TLR9) Cascade / protein lipidation / Synthesis of PIPs at the late endosome membrane / phosphatidylinositol 3-kinase complex, class III / Synthesis of PIPs at the early endosome membrane / cellular response to oxygen-glucose deprivation / phosphatidylinositol 3-kinase complex, class III, type II / positive regulation of stress granule assembly / phosphatidylinositol 3-kinase complex, class III, type I / mitochondria-associated endoplasmic reticulum membrane contact site / autophagy of peroxisome / response to mitochondrial depolarisation / positive regulation of attachment of mitotic spindle microtubules to kinetochore / cytoplasmic side of mitochondrial outer membrane / negative regulation of lysosome organization / positive regulation by host of viral genome replication / Synthesis of PIPs at the Golgi membrane / engulfment of apoptotic cell / positive regulation of autophagosome assembly / receptor catabolic process / negative regulation of autophagosome assembly / regulation of protein complex stability / phosphatidylinositol kinase activity / phosphatidylinositol 3-kinase regulator activity / protein localization to phagophore assembly site / suppression by virus of host autophagy / protein targeting to vacuole / protein targeting to lysosome / phagophore assembly site membrane / late endosome to vacuole transport / early endosome to late endosome transport / SMAD protein signal transduction / negative regulation of programmed cell death / Translation of Replicase and Assembly of the Replication Transcription Complex / phagophore assembly site / cellular response to nitrogen starvation / response to iron(II) ion / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / autophagosome membrane docking / cytoplasmic pattern recognition receptor signaling pathway / mitotic metaphase chromosome alignment / post-transcriptional regulation of gene expression / 1-phosphatidylinositol-3-kinase activity / endosome to lysosome transport / lysosome organization / Macroautophagy / RSV-host interactions / positive regulation of cardiac muscle hypertrophy / autolysosome / p38MAPK cascade / phosphatidylinositol-mediated signaling / axoneme / phosphatidylinositol phosphate biosynthetic process / autophagosome membrane / autophagosome maturation / autophagosome assembly / PI3K Cascade / mitophagy / RHO GTPases Activate NADPH Oxidases / response to vitamin E / negative regulation of reactive oxygen species metabolic process / neuron development / amyloid-beta metabolic process / regulation of macroautophagy / cellular defense response / phosphatidylinositol 3-kinase binding / cellular response to glucose starvation / positive regulation of autophagy / phagocytic vesicle / positive regulation of intrinsic apoptotic signaling pathway / protein-membrane adaptor activity / JNK cascade / cellular response to epidermal growth factor stimulus / cellular response to copper ion / autophagosome / phosphatidylinositol 3-kinase/protein kinase B signal transduction / cellular response to amino acid starvation / cellular response to starvation / regulation of cytokinesis / negative regulation of protein phosphorylation / regulation of autophagy / macroautophagy / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / response to lead ion / regulation of protein phosphorylation / trans-Golgi network / ISG15 antiviral mechanism / autophagy / cellular response to hydrogen peroxide / endocytosis / phagocytic vesicle membrane / microtubule cytoskeleton / late endosome Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 27.5 Å | |||||||||
Authors | Baskaran S / Carlson L-A / Stjepanovic G / Young LN / Kim DJ / Grob P / Stanley RE / Nogales E / Hurley JH | |||||||||
Citation | Journal: Elife / Year: 2014 Title: Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex. Authors: Sulochanadevi Baskaran / Lars-Anders Carlson / Goran Stjepanovic / Lindsey N Young / Do Jin Kim / Patricia Grob / Robin E Stanley / Eva Nogales / James H Hurley / Abstract: The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and ...The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2846.map.gz | 3.3 MB | EMDB map data format | |
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Header (meta data) | emd-2846-v30.xml emd-2846.xml | 15.3 KB 15.3 KB | Display Display | EMDB header |
Images | emd_2846.tif | 86.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2846 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2846 | HTTPS FTP |
-Validation report
Summary document | emd_2846_validation.pdf.gz | 199 KB | Display | EMDB validaton report |
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Full document | emd_2846_full_validation.pdf.gz | 198.1 KB | Display | |
Data in XML | emd_2846_validation.xml.gz | 5.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2846 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2846 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2846.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Three-dimensional structure of the autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.01 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
Entire | Name: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1. |
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Components |
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-Supramolecule #1000: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
Supramolecule | Name: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1. type: sample / ID: 1000 / Oligomeric state: heterotetramer / Number unique components: 4 |
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Molecular weight | Theoretical: 361.8 KDa |
-Macromolecule #1: phosphatidylinositol 3-kinase, catalytic subunit type 3
Macromolecule | Name: phosphatidylinositol 3-kinase, catalytic subunit type 3 type: protein_or_peptide / ID: 1 / Name.synonym: VPS34, PI3KC3 / Number of copies: 1 / Oligomeric state: one component of heterotetramer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane |
Molecular weight | Theoretical: 102 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG |
Sequence | UniProtKB: Phosphatidylinositol 3-kinase catalytic subunit type 3 GO: 1-phosphatidylinositol-3-kinase activity, phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly |
-Macromolecule #2: phosphoinositide-3-kinase, regulatory subunit 4
Macromolecule | Name: phosphoinositide-3-kinase, regulatory subunit 4 / type: protein_or_peptide / ID: 2 / Name.synonym: VPS15, p150, PIK3R4 / Number of copies: 1 / Oligomeric state: one component of heterotetramer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane |
Molecular weight | Theoretical: 153 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG |
Sequence | UniProtKB: Phosphoinositide 3-kinase regulatory subunit 4 GO: phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly |
-Macromolecule #3: beclin1
Macromolecule | Name: beclin1 / type: protein_or_peptide / ID: 3 / Name.synonym: BECN1 / Number of copies: 1 / Oligomeric state: one component of heterotetramer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane |
Molecular weight | Theoretical: 52 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG |
Sequence | UniProtKB: Beclin-1 GO: phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly |
-Macromolecule #4: autophagy related 14
Macromolecule | Name: autophagy related 14 / type: protein_or_peptide / ID: 4 / Name.synonym: ATG14, ATG14L, BARKOR / Number of copies: 1 / Oligomeric state: one component of heterotetramer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane |
Molecular weight | Theoretical: 55 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG |
Sequence | UniProtKB: Beclin 1-associated autophagy-related key regulator GO: phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 8 Details: 20 mM Tris-HCl, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 3% trehalose |
Staining | Type: NEGATIVE Details: Protein was incubated on grids for 30s, followed by sequential 10 s incubations on four 50 ul drops of 1% uranyl formate. The stained grids were blotted to near dryness with a filter paper and air-dried. |
Grid | Details: Continuous carbon, plasma cleaned for 10 s in a Gatan Solarus at 10% O2. |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Min: 292 K / Max: 294 K / Average: 293 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 80,000 times magnification. |
Date | May 28, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 1614 / Average electron dose: 25 e/Å2 / Bits/pixel: 32 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 100333 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 45 |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | The final three-dimensional structure was calculated in RELION using a single 3D class. Starting model for this calculation was a random conical tilt model low-pass filtered to 80A. |
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CTF correction | Details: tilt-dependent phase flip |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 27.5 Å / Resolution method: OTHER / Software - Name: Spider, EMAN, EMAN2, IMAGIC, RELION Details: Final map calculated in RELION using a single 3D class. Data acquired at 0, 30, and 45 degrees were used to refine an initial RCT model low-pass filtered to 80A. Gold standard FSC calculated using RELION. Number images used: 38745 |