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- PDB-9rx6: VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RA... -

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Basic information

Entry
Database: PDB / ID: 9rx6
TitleVPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RAB5A (Q79L) on the VPS15 subunit
Components
  • Beclin-1
  • Phosphatidylinositol 3-kinase catalytic subunit type 3
  • Phosphoinositide 3-kinase regulatory subunit 4
  • Ras-related protein Rab-5A
  • UV radiation resistance associated protein
KeywordsENDOCYTOSIS / Lipid kinase / GTPase / kinase / endocytic sorting / endosome maturation / SIGNALING PROTEIN
Function / homology
Function and homology information


protein-containing complex organization / regulation of endosome size / nucleus-vacuole junction / cytoplasmic side of early endosome membrane / cellular response to aluminum ion / positive regulation of protein lipidation / protein localization to early endosome / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / positive regulation of stress granule assembly ...protein-containing complex organization / regulation of endosome size / nucleus-vacuole junction / cytoplasmic side of early endosome membrane / cellular response to aluminum ion / positive regulation of protein lipidation / protein localization to early endosome / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / positive regulation of stress granule assembly / Synthesis of PIPs at the late endosome membrane / phosphatidylinositol 3-kinase complex, class III / 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 / amyloid-beta clearance by transcytosis / response to mitochondrial depolarisation / presynaptic endosome / positive regulation of attachment of mitotic spindle microtubules to kinetochore / host-mediated activation of viral genome replication / synaptic vesicle recycling / plasma membrane to endosome transport / Dengue virus modulates apoptosis / engulfment of apoptotic cell / negative regulation of lysosome organization / host-mediated perturbation of viral process / phosphatidylinositol kinase activity / SMAD protein signal transduction / positive regulation of autophagosome assembly / regulation of filopodium assembly / Synthesis of PIPs at the Golgi membrane / cytoplasmic side of mitochondrial outer membrane / early endosome to late endosome transport / receptor catabolic process / RAB geranylgeranylation / response to L-leucine / protein targeting to vacuole / late endosome to vacuole transport / regulation of autophagosome assembly / protein targeting to lysosome / endosome organization / pexophagy / RAB GEFs exchange GTP for GDP on RABs / early phagosome / positive regulation of natural killer cell mediated cytotoxicity / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / TBC/RABGAPs / cellular response to nitrogen starvation / centrosome cycle / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / endosomal transport / 1-phosphatidylinositol-3-kinase activity / negative regulation of programmed cell death / response to vitamin E / Macroautophagy / response to iron(II) ion / regulation of synaptic vesicle exocytosis / RSV-host interactions / p38MAPK cascade / cytoplasmic pattern recognition receptor signaling pathway / phosphatidylinositol phosphate biosynthetic process / Synthesis of PIPs at the plasma membrane / mitotic metaphase chromosome alignment / phosphatidylinositol-mediated signaling / autolysosome / positive regulation of exocytosis / Respiratory syncytial virus (RSV) attachment and entry / canonical Wnt signaling pathway / PI3K Cascade / chromosome, centromeric region / RHO GTPases Activate NADPH Oxidases / regulation of macroautophagy / autophagosome maturation / endocytic vesicle / axoneme / synaptic vesicle endocytosis / cellular defense response / autophagosome assembly / phagocytosis / phosphatidylinositol 3-kinase binding / cellular response to glucose starvation / intercellular bridge / mitophagy / JNK cascade / phagocytic vesicle / ruffle / positive regulation of intrinsic apoptotic signaling pathway / vesicle-mediated transport / somatodendritic compartment / endomembrane system / positive regulation of autophagy / Prevention of phagosomal-lysosomal fusion / autophagosome / cellular response to epidermal growth factor stimulus / axon terminus / cellular response to copper ion / cellular response to amino acid starvation
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 / : / : / PIK3R4-like, middle domain / HEAT, type 2 / HEAT repeat profile. / Small GTPase Rab domain profile. / 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. / Protein kinase C conserved region 2 (CalB) / C2 domain / 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 / WD domain, G-beta repeat / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Trp-Asp (WD) repeats signature. / Protein kinase domain / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / Serine/Threonine protein kinases, catalytic domain / WD40-repeat-containing domain superfamily / 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
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / MYRISTIC ACID / Ras-related protein Rab-5A / Beclin-1 / UV radiation resistance associated protein / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Phosphoinositide 3-kinase regulatory subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsSpokaite, S. / Ohashi, Y. / Dessus, A.N. / Bourguet, M. / Williams, R.L.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105184308 United Kingdom
Cancer Research UKDRCPGM 100014 United Kingdom
Citation
Journal: Elife / Year: 2026
Title: A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution.
Authors: Saule Spokaite / Yohei Ohashi / Maxime Bourguet / Antoine Nicolas Dessus / Roger L Williams /
Abstract: RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by ...RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in VPS15 resulted in defective CPY sorting, loss of colocalisation with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.
#1: Journal: Elife / Year: 2026
Title: A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution
Authors: Spokaite, S. / Ohashi, Y. / Bourguet, M. / Dessus, A.N. / Williams, R.L.
History
DepositionJul 10, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 10, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update
Revision 1.1Jun 10, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin / Data content type: EM metadata / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphatidylinositol 3-kinase catalytic subunit type 3
B: Phosphoinositide 3-kinase regulatory subunit 4
C: Beclin-1
D: UV radiation resistance associated protein
F: Ras-related protein Rab-5A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)411,25311
Polymers409,9445
Non-polymers1,3096
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 5 types, 5 molecules ABCDF

#1: Protein Phosphatidylinositol 3-kinase catalytic subunit type 3 / PI3-kinase type 3 / PI3K type 3 / PtdIns-3-kinase type 3 / Phosphatidylinositol 3-kinase p100 ...PI3-kinase type 3 / PI3K type 3 / PtdIns-3-kinase type 3 / Phosphatidylinositol 3-kinase p100 subunit / Phosphoinositide-3-kinase class 3 / hVps34


Mass: 101708.406 Da / Num. of mol.: 1 / Mutation: 199-REIE-202 to 199-ERIR-202
Source method: isolated from a genetically manipulated source
Details: Full length VPS34 with a 199-REIE-202 to 199-ERIR-202 mutation
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3C3, VPS34 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q8NEB9, phosphatidylinositol 3-kinase
#2: Protein Phosphoinositide 3-kinase regulatory subunit 4 / PI3-kinase regulatory subunit 4 / PI3-kinase p150 subunit / Phosphoinositide 3-kinase adaptor protein


Mass: 154659.188 Da / Num. of mol.: 1 / Mutation: Extra SRPTTASENLYFQ at C-terminus
Source method: isolated from a genetically manipulated source
Details: Full length VPS15 myristoylated at the G2 residue, bound to GDP, with 13 extra residues at the C-terminus left after TEV protease treatment (VPS15-SRPTTASENLYFQ)
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3R4, VPS15 / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: Q99570, non-specific serine/threonine protein kinase
#3: Protein Beclin-1 / Coiled-coil myosin-like BCL2-interacting protein / Protein GT197


Mass: 51953.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BECN1, GT197 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q14457
#4: Protein UV radiation resistance associated protein


Mass: 78289.852 Da / Num. of mol.: 1 / Mutation: P10Q
Source method: isolated from a genetically manipulated source
Details: Full length UVRAG with a P10Q mutation / Source: (gene. exp.) Homo sapiens (human) / Gene: UVRAG / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q6P1X0
#5: Protein Ras-related protein Rab-5A


Mass: 23333.225 Da / Num. of mol.: 1 / Mutation: C19S, C63S, Q79L
Source method: isolated from a genetically manipulated source
Details: RAB5A-GTP (1-212) C19S C63S Q79L GTP-locked mutant / Source: (gene. exp.) Homo sapiens (human) / Gene: RAB5A, RAB5 / Production host: Escherichia coli (E. coli) / Strain (production host): OverExpress(TM) C41(DE3) / References: UniProt: P20339, small monomeric GTPase

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Non-polymers , 5 types, 6 molecules

#6: Chemical ChemComp-MYR / MYRISTIC ACID


Mass: 228.371 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H28O2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationY

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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: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
Type: COMPLEX / Entity ID: #5, #3-#4, #1 / Source: RECOMBINANT
Molecular weightValue: 0.409 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2200 mMSodium chlorideNaCl1
31 mMTCEPC9H15O6P1
45 mMMagnesium chlorideMgCl21
51 mMAMPPNPC10H17N6O12P31
64 mMCHAPSOC32H58N2O8S1
70.005 % v/vNonidet P40 substitute(C2H4O)nC14H22O1
SpecimenConc.: 2.45 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was crosslinked with 30 uM BS3 for 20 minutes on ice, then quenched with 100 mM TRIS pH 8.0 prior to vitrification
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 287 K
Details: 3.5 uL sample, 0 second wait, 3.5 second blot, blot force +8

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13301
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7.5particle selection
2PHENIX1.21rc1_5156:model refinement
13cryoSPARC3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 370889
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84296 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00424556
ELECTRON MICROSCOPYf_angle_d0.70833198
ELECTRON MICROSCOPYf_dihedral_angle_d8.0163270
ELECTRON MICROSCOPYf_chiral_restr0.0463686
ELECTRON MICROSCOPYf_plane_restr0.0064259

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