+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12214 | ||||||||||||||||||||||||
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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 |
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Keywords | Rab GTPase / Phosphatidylinositol 3-kinase / ENDOCYTOSIS | ||||||||||||||||||||||||
Function / homology | Function and homology information 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 / synaptic vesicle recycling / 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 / autophagy of peroxisome / 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 / negative regulation of lysosome organization / modulation by host of viral process / 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 / phosphatidylinositol kinase activity / multivesicular body sorting pathway / protein localization to phagophore assembly site / suppression by virus of host autophagy / protein targeting to vacuole / regulation of filopodium assembly / protein targeting to lysosome / RAB geranylgeranylation / late endosome to vacuole transport / early endosome to late endosome transport / SMAD protein signal transduction / regulation of autophagosome assembly / RAB GEFs exchange GTP for GDP on RABs / double-strand break repair via classical nonhomologous end joining / early phagosome / negative regulation of programmed cell death / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / Translation of Replicase and Assembly of the Replication Transcription Complex / phagophore assembly site / centrosome cycle / positive regulation of autophagosome maturation / cellular response to nitrogen starvation / response to iron(II) ion / TBC/RABGAPs / phosphatidylinositol 3-kinase / SNARE complex assembly / phosphatidylinositol-3-phosphate biosynthetic process / spindle organization / regulation of synaptic vesicle exocytosis / cytoplasmic pattern recognition receptor signaling pathway / mitotic metaphase chromosome alignment / 1-phosphatidylinositol-3-kinase activity / lysosome organization / Macroautophagy / RSV-host interactions / positive regulation of cardiac muscle hypertrophy / autolysosome / p38MAPK cascade / positive regulation of exocytosis / Synthesis of PIPs at the plasma membrane / phosphatidylinositol-mediated signaling / axoneme / phosphatidylinositol phosphate biosynthetic process / Respiratory syncytial virus (RSV) attachment and entry / autophagosome membrane / autophagosome maturation / autophagosome assembly / chromosome, centromeric region / PI3K Cascade / mitophagy / RHO GTPases Activate NADPH Oxidases / response to vitamin E / negative regulation of reactive oxygen species metabolic process / canonical Wnt signaling pathway / neuron development / amyloid-beta metabolic process / regulation of macroautophagy / cellular defense response / endomembrane system / phosphatidylinositol 3-kinase binding / cellular response to glucose starvation / phagocytosis / positive regulation of autophagy / phagocytic vesicle / positive regulation of intrinsic apoptotic signaling pathway / axon terminus / JNK cascade / ruffle / cellular response to epidermal growth factor stimulus Similarity search - Function | ||||||||||||||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||||||||||||||
Method | subtomogram averaging / cryo EM / Resolution: 9.8 Å | ||||||||||||||||||||||||
Authors | Tremel S / Morado DR | ||||||||||||||||||||||||
Funding support | United Kingdom, 7 items
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Citation | 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 |
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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_12214.map.gz | 119.6 MB | EMDB map data format | |
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Header (meta data) | emd-12214-v30.xml emd-12214.xml | 34.1 KB 34.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12214_fsc.xml | 5.8 KB | Display | FSC data file |
Images | emd_12214.png | 142.5 KB | ||
Filedesc metadata | emd-12214.cif.gz | 10.1 KB | ||
Others | emd_12214_half_map_1.map.gz emd_12214_half_map_2.map.gz | 2.5 MB 2.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12214 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12214 | HTTPS FTP |
-Validation report
Summary document | emd_12214_validation.pdf.gz | 485 KB | Display | EMDB validaton report |
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Full document | emd_12214_full_validation.pdf.gz | 484.6 KB | Display | |
Data in XML | emd_12214_validation.xml.gz | 12.5 KB | Display | |
Data in CIF | emd_12214_validation.cif.gz | 16.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12214 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12214 | HTTPS FTP |
-Related structure data
Related structure data | 7bl1MC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12214.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.0665 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: Odd Half-map masked to remove density from the membrane.
File | emd_12214_half_map_1.map | ||||||||||||
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Annotation | Odd Half-map masked to remove density from the membrane. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Even Half-map masked to remove density from the membrane.
File | emd_12214_half_map_2.map | ||||||||||||
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Annotation | Even Half-map masked to remove density from the membrane. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...
+Supramolecule #1: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...
+Supramolecule #2: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)
+Supramolecule #3: Ras-related protein Rab-5A
+Macromolecule #1: UV radiation resistance-associated gene protein
+Macromolecule #2: Phosphatidylinositol 3-kinase catalytic subunit type 3
+Macromolecule #3: Phosphoinositide 3-kinase regulatory subunit 4
+Macromolecule #4: Beclin-1
+Macromolecule #5: unknown peptide
+Macromolecule #6: Ras-related protein Rab-5A
+Macromolecule #7: GUANOSINE-5'-TRIPHOSPHATE
+Macromolecule #8: MAGNESIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | 3D array |
-Sample preparation
Concentration | 6.2 mg/mL | ||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: Quorum SC7620 | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 316 K / Instrument: FEI VITROBOT MARK I / Details: Blot force 20, blot time 6 s. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Average exposure time: 0.55 sec. / Average electron dose: 2.99 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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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: |