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- PDB-5kc2: Negative stain structure of Vps15/Vps34 complex -

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Basic information

Entry
Database: PDB / ID: 5kc2
TitleNegative stain structure of Vps15/Vps34 complex
Components
  • Phosphatidylinositol 3-kinase VPS34
  • Serine/threonine-protein kinase VPS15
KeywordsENDOCYTOSIS / autophagy / phosphatidylinositol 3-kinase (PtdIns3K)
Function / homology
Function and homology information


Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the early endosome membrane / RHO GTPases Activate NADPH Oxidases / autophagy of peroxisome / nucleus-vacuole junction / Synthesis of PIPs at the Golgi membrane / vacuole-isolation membrane contact site / vacuole inheritance / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I ...Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the early endosome membrane / RHO GTPases Activate NADPH Oxidases / autophagy of peroxisome / nucleus-vacuole junction / Synthesis of PIPs at the Golgi membrane / vacuole-isolation membrane contact site / vacuole inheritance / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I / Macroautophagy / protein retention in Golgi apparatus / pexophagy / phagophore assembly site membrane / protein targeting to vacuole / late endosome to vacuole transport / fungal-type vacuole membrane / phagophore assembly site / phosphatidylinositol-mediated signaling / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / phosphatidylinositol phosphate biosynthetic process / autophagosome assembly / ubiquitin binding / positive regulation of transcription elongation by RNA polymerase II / macroautophagy / autophagy / peroxisome / endocytosis / protein transport / late endosome / endosome membrane / non-specific serine/threonine protein kinase / endosome / protein kinase activity / phosphorylation / Golgi membrane / protein serine kinase activity / protein serine/threonine kinase activity / mitochondrion / ATP binding / membrane / cytosol / cytoplasm
Similarity search - Function
Serine/threonine-protein kinase Vps15-like / Phosphatidylinositol 3-kinase, Vps34 type / HEAT repeat profile. / HEAT, type 2 / 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) ...Serine/threonine-protein kinase Vps15-like / Phosphatidylinositol 3-kinase, Vps34 type / HEAT repeat profile. / HEAT, type 2 / 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-like helical / Armadillo-type fold / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / WD40-repeat-containing domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine/threonine-protein kinase VPS15 / Phosphatidylinositol 3-kinase VPS34
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 28 Å
AuthorsKirsten, M.L. / Zhang, L. / Ohashi, Y. / Perisic, O. / Williams, R.L. / Sachse, C.
CitationJournal: Autophagy / Year: 2016
Title: Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex.
Authors: Yohei Ohashi / Nicolas Soler / Miguel García Ortegón / Lufei Zhang / Marie L Kirsten / Olga Perisic / Glenn R Masson / John E Burke / Arjen J Jakobi / Apostolos A Apostolakis / Christopher ...Authors: Yohei Ohashi / Nicolas Soler / Miguel García Ortegón / Lufei Zhang / Marie L Kirsten / Olga Perisic / Glenn R Masson / John E Burke / Arjen J Jakobi / Apostolos A Apostolakis / Christopher M Johnson / Maki Ohashi / Nicholas T Ktistakis / Carsten Sachse / Roger L Williams /
Abstract: The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization ...The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.
History
DepositionJun 4, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Oct 5, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2016Group: Database references
Revision 1.2Nov 9, 2016Group: Database references
Revision 1.3Jul 26, 2017Group: Experimental preparation / Category: em_sample_support
Revision 1.4Aug 2, 2017Group: Data collection / Category: em_software / Item: _em_software.name / _em_software.version
Revision 1.5Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.6Nov 13, 2019Group: Data collection / Other / Category: symmetry
Item: _symmetry.Int_Tables_number / _symmetry.space_group_name_H-M

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Structure visualization

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Assembly

Deposited unit
C: Phosphatidylinositol 3-kinase VPS34
B: Serine/threonine-protein kinase VPS15


Theoretical massNumber of molelcules
Total (without water)267,5972
Polymers267,5972
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phosphatidylinositol 3-kinase VPS34 / PtdIns-3-kinase VPS34 / Carboxypeptidase Y-deficient protein 15 / Vacuolar protein sorting- ...PtdIns-3-kinase VPS34 / Carboxypeptidase Y-deficient protein 15 / Vacuolar protein sorting-associated protein 34 / Vacuolar protein-targeting protein 29


Mass: 101047.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: VPS34, END12, PEP15, VPL7, VPT29, YLR240W, L9672.10 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P22543, phosphatidylinositol 3-kinase
#2: Protein Serine/threonine-protein kinase VPS15 / Golgi-retention defective mutant protein 8 / Vacuolar protein sorting-associated protein 15


Mass: 166550.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: VPS15, GRD8, VAC4, VPL19, YBR097W, YBR0825 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P22219, non-specific serine/threonine protein kinase

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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: Vps15/34 / Type: COMPLEX
Details: Subcomplex of Vps34 phosphatidylinositol 3-kinase (PtdIns3K) complex I (yeast)
Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: pYO225
Buffer solutionpH: 8.8
Buffer component
IDConc.NameBuffer-ID
120 mMTris1
2500 mMNaClSodium chloride1
31 %CHAPS1
42 mMDTT1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Details: droplet technique / Material: Uranyl acetate
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid type
11COPPER300Plano 300 mesh
21COPPER300Quantifoil R2/2

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 1 sec. / Electron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Num. of grids imaged: 2

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
4EMAN2CTF correction
10SPIDERinitial Euler assignment
11SPIDERfinal Euler assignment
12IMAGICclassification
13SPIDER3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 14172
3D reconstructionResolution: 28 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 14172 / Algorithm: BACK PROJECTION / Symmetry type: POINT

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