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- EMDB-2846: Three-dimensional structure of the autophagic phosphatidylinosito... -

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Entry
Database: EMDB / ID: EMD-2846
TitleThree-dimensional structure of the autophagic phosphatidylinositol 3-kinase complex.
Map dataThree-dimensional structure of the autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
Sample
  • Sample: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
  • Protein or peptide: phosphatidylinositol 3-kinase, catalytic subunit type 3
  • Protein or peptide: phosphoinositide-3-kinase, regulatory subunit 4
  • Protein or peptide: beclin1
  • Protein or peptide: autophagy related 14
Keywordskinase / 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 / postsynaptic endosome / 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 / postsynaptic endosome / Synthesis of PIPs at the late endosome membrane / phosphatidylinositol 3-kinase complex, class III / engulfment of apoptotic cell / cellular response to oxygen-glucose deprivation / Synthesis of PIPs at the early endosome membrane / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I / presynaptic endosome / positive regulation of stress granule assembly / response to mitochondrial depolarisation / positive regulation of protein lipidation / positive regulation by host of viral genome replication / positive regulation of attachment of mitotic spindle microtubules to kinetochore / mitochondria-associated endoplasmic reticulum membrane contact site / negative regulation of lysosome organization / Synthesis of PIPs at the Golgi membrane / phosphatidylinositol kinase activity / regulation of protein complex stability / positive regulation of autophagosome assembly / phosphatidylinositol 3-kinase regulator activity / negative regulation of autophagosome assembly / cytoplasmic side of mitochondrial outer membrane / protein localization to phagophore assembly site / receptor catabolic process / phagophore assembly site membrane / protein targeting to vacuole / SMAD protein signal transduction / protein targeting to lysosome / early endosome to late endosome transport / late endosome to vacuole transport / pexophagy / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / phosphatidylinositol-3-phosphate biosynthetic process / cellular response to nitrogen starvation / negative regulation of programmed cell death / response to iron(II) ion / phosphatidylinositol 3-kinase / lysosome organization / 1-phosphatidylinositol-3-kinase activity / mitotic metaphase chromosome alignment / post-transcriptional regulation of gene expression / cytoplasmic pattern recognition receptor signaling pathway / autophagosome membrane docking / endosome to lysosome transport / Macroautophagy / phosphatidylinositol-mediated signaling / positive regulation of cardiac muscle hypertrophy / RSV-host interactions / p38MAPK cascade / phosphatidylinositol phosphate biosynthetic process / autolysosome / regulation of protein phosphorylation / autophagosome membrane / negative regulation of protein phosphorylation / synaptic vesicle endocytosis / PI3K Cascade / axoneme / autophagosome assembly / RHO GTPases Activate NADPH Oxidases / response to vitamin E / autophagosome maturation / amyloid-beta metabolic process / regulation of macroautophagy / neuron development / phosphatidylinositol 3-kinase binding / cellular defense response / cellular response to glucose starvation / mitophagy / positive regulation of intrinsic apoptotic signaling pathway / protein-membrane adaptor activity / phagocytic vesicle / JNK cascade / cellular response to copper ion / positive regulation of autophagy / cellular response to epidermal growth factor stimulus / cellular response to amino acid starvation / autophagosome / cellular response to starvation / regulation of cytokinesis / GABA-ergic synapse / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / phosphatidylinositol 3-kinase/protein kinase B signal transduction / macroautophagy / trans-Golgi network / response to lead ion / ISG15 antiviral mechanism / autophagy / cellular response to hydrogen peroxide / phagocytic vesicle membrane / circadian rhythm / endocytosis
Similarity search - Function
UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / : / VPS15-like, helical domain / Beclin-1, BH3 domain / Beclin-1 BH3 domain, Bcl-2-interacting / Atg6/Beclin / Atg6/Beclin C-terminal domain superfamily / Atg6, BARA domain ...UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / : / VPS15-like, helical domain / Beclin-1, BH3 domain / Beclin-1 BH3 domain, Bcl-2-interacting / Atg6/Beclin / Atg6/Beclin C-terminal domain superfamily / Atg6, BARA domain / Atg6/beclin, coiled-coil domain / Apg6 BARA domain / Apg6 coiled-coil region / Phosphatidylinositol 3-kinase, Vps34 type / HEAT repeat profile. / HEAT, type 2 / C2 phosphatidylinositol 3-kinase-type domain / Phosphoinositide 3-kinase C2 / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, region postulated to contain C2 domain / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / 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-like helical / Armadillo-type fold / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40 repeat / WD40-repeat-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Beclin-1 / Beclin 1-associated autophagy-related key regulator / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Phosphoinositide 3-kinase regulatory subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 27.5 Å
AuthorsBaskaran S / Carlson L-A / Stjepanovic G / Young LN / Kim DJ / Grob P / Stanley RE / Nogales E / Hurley JH
CitationJournal: 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
DepositionDec 26, 2014-
Header (metadata) releaseJan 14, 2015-
Map releaseJan 14, 2015-
UpdateJan 14, 2015-
Current statusJan 14, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2846.tif

Downloads & links

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Map

FileDownload / File: emd_2846.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThree-dimensional structure of the autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.01 Å/pix.
x 128 pix.
= 385.28 Å		3.01 Å/pix.
x 128 pix.
= 385.28 Å		3.01 Å/pix.
x 128 pix.
= 385.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.01 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.02966253 - 0.09436484
Average (Standard dev.)0.00045778 (±0.00435199)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 385.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.013.013.01
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z385.280385.280385.280
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0300.0940.000

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Supplemental data

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Sample components

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Entire : Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.

EntireName: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
Components
  • Sample: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
  • Protein or peptide: phosphatidylinositol 3-kinase, catalytic subunit type 3
  • Protein or peptide: phosphoinositide-3-kinase, regulatory subunit 4
  • Protein or peptide: beclin1
  • Protein or peptide: autophagy related 14

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Supramolecule #1000: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.

SupramoleculeName: Autophagic phosphatidylinositol 3-kinase complex, PI3KC3-C1.
type: sample / ID: 1000 / Oligomeric state: heterotetramer / Number unique components: 4
Molecular weightTheoretical: 361.8 KDa

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Macromolecule #1: phosphatidylinositol 3-kinase, catalytic subunit type 3

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane
Molecular weightTheoretical: 102 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG
SequenceUniProtKB: Phosphatidylinositol 3-kinase catalytic subunit type 3
GO: 1-phosphatidylinositol-3-kinase activity, phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly

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Macromolecule #2: phosphoinositide-3-kinase, regulatory subunit 4

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane
Molecular weightTheoretical: 153 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG
SequenceUniProtKB: Phosphoinositide 3-kinase regulatory subunit 4
GO: phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly

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Macromolecule #3: beclin1

MacromoleculeName: beclin1 / type: protein_or_peptide / ID: 3 / Name.synonym: BECN1 / Number of copies: 1 / Oligomeric state: one component of heterotetramer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane
Molecular weightTheoretical: 52 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG
SequenceUniProtKB: Beclin-1
GO: phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly

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Macromolecule #4: autophagy related 14

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: autophagosome / Location in cell: autophagosome membrane
Molecular weightTheoretical: 55 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pCAG
SequenceUniProtKB: Beclin 1-associated autophagy-related key regulator
GO: phosphatidylinositol 3-kinase complex, class III, type I, autophagy, autophagosome assembly

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 8
Details: 20 mM Tris-HCl, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 3% trehalose
StainingType: 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.
GridDetails: Continuous carbon, plasma cleaned for 10 s in a Gatan Solarus at 10% O2.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 292 K / Max: 294 K / Average: 293 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 80,000 times magnification.
DateMay 28, 2014
Image recordingCategory: 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 min0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 45
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe 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.
CTF correctionDetails: tilt-dependent phase flip
Final reconstructionApplied 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

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