- EMDB-12214: human complex II-BATS bound to membrane-attached Rab5a-GTP -
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Basic information
Entry
Database: EMDB / ID: EMD-12214
Title
human complex II-BATS bound to membrane-attached Rab5a-GTP
Map data
Subtomogram averaging reconstruction of human complex II-BATS bound to membrane-attached Rab5a-GTP at 9.8A with the membrane masked out after local-denoising with the LAFTER algorithm
Sample
Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)
Protein or peptide: UV radiation resistance-associated gene protein
Protein or peptide: Phosphatidylinositol 3-kinase catalytic subunit type 3
Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 4
lytic vacuole / maintenance of Golgi location / regulation of protein serine/threonine kinase activity / regulation of endosome size / nucleus-vacuole junction / cellular response to aluminum ion / postsynaptic early endosome / cytoplasmic side of early endosome membrane / Toll Like Receptor 9 (TLR9) Cascade / protein lipidation ...lytic vacuole / maintenance of Golgi location / regulation of protein serine/threonine kinase activity / regulation of endosome size / nucleus-vacuole junction / cellular response to aluminum ion / postsynaptic early endosome / cytoplasmic side of early endosome membrane / 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 / synaptic vesicle recycling / phosphatidylinositol 3-kinase complex, class III, type II / positive regulation of stress granule assembly / cellular response to oxygen-glucose deprivation / phosphatidylinositol 3-kinase complex, class III, type I / response to mitochondrial depolarisation / positive regulation of attachment of mitotic spindle microtubules to kinetochore / amyloid-beta clearance by transcytosis / cytoplasmic side of mitochondrial outer membrane / positive regulation by host of viral genome replication / negative regulation of lysosome organization / modulation by host of viral process / Synthesis of PIPs at the Golgi membrane / engulfment of apoptotic cell / negative regulation of autophagosome assembly / positive regulation of autophagosome assembly / phosphatidylinositol kinase activity / receptor catabolic process / protein targeting to vacuole / protein localization to phagophore assembly site / suppression by virus of host autophagy / multivesicular body sorting pathway / RAB geranylgeranylation / regulation of filopodium assembly / protein targeting to lysosome / early endosome to late endosome transport / late endosome to vacuole transport / regulation of autophagosome assembly / SMAD protein signal transduction / RAB GEFs exchange GTP for GDP on RABs / double-strand break repair via classical nonhomologous end joining / early phagosome / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / Respiratory syncytial virus (RSV) attachment and entry / Translation of Replicase and Assembly of the Replication Transcription Complex / phagophore assembly site / negative regulation of programmed cell death / cellular response to nitrogen starvation / response to iron(II) ion / TBC/RABGAPs / centrosome cycle / positive regulation of autophagosome maturation / SNARE complex assembly / autolysosome / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / spindle organization / regulation of synaptic vesicle exocytosis / mitotic metaphase chromosome alignment / cytoplasmic pattern recognition receptor signaling pathway / 1-phosphatidylinositol-3-kinase activity / Macroautophagy / lysosome organization / RSV-host interactions / positive regulation of cardiac muscle hypertrophy / p38MAPK cascade / positive regulation of exocytosis / axoneme / Synthesis of PIPs at the plasma membrane / phosphatidylinositol-mediated signaling / phosphatidylinositol phosphate biosynthetic process / autophagosome membrane / autophagosome maturation / cellular response to epidermal growth factor stimulus / chromosome, centromeric region / mitophagy / autophagosome assembly / autophagosome / PI3K Cascade / RHO GTPases Activate NADPH Oxidases / negative regulation of reactive oxygen species metabolic process / canonical Wnt signaling pathway / response to vitamin E / neuron development / endomembrane system / amyloid-beta metabolic process / regulation of macroautophagy / cellular defense response / phosphatidylinositol 3-kinase binding / positive regulation of autophagy / phagocytosis / cellular response to glucose starvation / positive regulation of intrinsic apoptotic signaling pathway / axon terminus / JNK cascade / phagocytic vesicle / ruffle 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 / 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 ...UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / 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 / small GTPase Rab1 family 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. / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 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 / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / C2 domain superfamily / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40-repeat-containing domain superfamily / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase Similarity search - Domain/homology
Ras-related protein Rab-5A / Beclin-1 / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Phosphoinositide 3-kinase regulatory subunit 4 / UV radiation resistance-associated gene protein Similarity search - Component
Biological species
Homo sapiens (human)
Method
subtomogram averaging / cryo EM / Resolution: 9.8 Å
Journal: Nat Commun / Year: 2021 Title: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes. Authors: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel ...Authors: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel / Juri Rappsilber / Kathrin Lang / Sean Munro / John A G Briggs / Roger L Williams / Abstract: The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal ...The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
History
Deposition
Jan 17, 2021
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Header (metadata) release
Mar 3, 2021
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Map release
Mar 3, 2021
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Update
May 1, 2024
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Current status
May 1, 2024
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_12214.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Subtomogram averaging reconstruction of human complex II-BATS bound to membrane-attached Rab5a-GTP at 9.8A with the membrane masked out after local-denoising with the LAFTER algorithm
Voxel size
X=Y=Z: 1.0665 Å
Density
Contour Level
By AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum
-0.045468677 - 0.06802449
Average (Standard dev.)
0.00014741789 (±0.002034834)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
320
320
320
Spacing
320
320
320
Cell
A=B=C: 341.27997 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.0665
1.0665
1.0665
M x/y/z
320
320
320
origin x/y/z
0.000
0.000
0.000
length x/y/z
341.280
341.280
341.280
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
320
320
320
D min/max/mean
-0.045
0.068
0.000
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Supplemental data
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Half map: Odd Half-map masked to remove density from the membrane.
Entire : human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...
Entire
Name: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
Components
Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)
Protein or peptide: UV radiation resistance-associated gene protein
Protein or peptide: Phosphatidylinositol 3-kinase catalytic subunit type 3
Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 4
Protein or peptide: Beclin-1
Protein or peptide: unknown peptide
Complex: Ras-related protein Rab-5A
Protein or peptide: Ras-related protein Rab-5A
Ligand: GUANOSINE-5'-TRIPHOSPHATE
Ligand: MAGNESIUM ION
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Supramolecule #1: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...
Supramolecule
Name: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6 Details: The subunits were expressed using transient transfection in HEK293T cells. Cells were transfected with three plasmids: pYO1025 (encoding VPS34 and VPS15 in a pCAG backbone), pYO1124 ...Details: The subunits were expressed using transient transfection in HEK293T cells. Cells were transfected with three plasmids: pYO1025 (encoding VPS34 and VPS15 in a pCAG backbone), pYO1124 (encoding UVGRAG 1-464 fused to the BATS of ATG14, residues 413-492 in pVAG) and pYO1006 (Beclin1 in pCAG)
Molecular weight
Theoretical: 392615 kDa/nm
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Supramolecule #2: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)
Supramolecule
Name: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5
chain_id: A, source_name: PDB, initial_model_type: experimental model
Details
An initial model was build with SWISSMODEL. using 5DFZ, 4DDP and 3IHY. This was then fit manually to density while running restrained MD with ISOLDE. The model was refined in REFMAC with self-restraints (4.3) and restraints to 3MJH, 4DDP and 3IHY.
Refinement
Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 320 / Target criteria: correlation
Output model
PDB-7bl1: human complex II-BATS bound to membrane-attached Rab5a-GTP
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