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- PDB-9rx4: VPS34-CI bound to NRBF2 and RAB1A -

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Basic information

Entry
Database: PDB / ID: 9rx4
TitleVPS34-CI bound to NRBF2 and RAB1A
Components
  • Beclin 1-associated autophagy-related key regulator
  • Beclin-1
  • Nuclear receptor-binding factor 2
  • Phosphatidylinositol 3-kinase catalytic subunit type 3
  • Phosphoinositide 3-kinase regulatory subunit 4
  • Ras-related protein Rab-1A
KeywordsSIGNALING PROTEIN / Lipid kinase / GTPase / kinase / autophagy
Function / homology
Function and homology information


extrinsic component of omegasome membrane / positive regulation of glycoprotein metabolic process / phosphatidylinositol 3-kinase inhibitor activity / extrinsic component of phagophore assembly site membrane / growth hormone secretion / nucleus-vacuole junction / cellular response to aluminum ion / positive regulation of protein lipidation / positive regulation of stress granule assembly / postsynaptic endosome ...extrinsic component of omegasome membrane / positive regulation of glycoprotein metabolic process / phosphatidylinositol 3-kinase inhibitor activity / extrinsic component of phagophore assembly site membrane / growth hormone secretion / nucleus-vacuole junction / cellular response to aluminum ion / positive regulation of protein lipidation / positive regulation of stress granule assembly / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / 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 / response to mitochondrial depolarisation / presynaptic endosome / positive regulation of attachment of mitotic spindle microtubules to kinetochore / host-mediated activation of viral genome replication / COPII-coated vesicle cargo loading / mitochondria-associated endoplasmic reticulum membrane contact site / engulfment of apoptotic cell / regulation of protein complex stability / negative regulation of lysosome organization / phosphatidylinositol kinase activity / SMAD protein signal transduction / positive regulation of autophagosome assembly / phosphatidylinositol 3-kinase regulator activity / Synthesis of PIPs at the Golgi membrane / cytoplasmic side of mitochondrial outer membrane / early endosome to late endosome transport / phagophore assembly site membrane / receptor catabolic process / melanosome transport / RAB geranylgeranylation / response to L-leucine / protein targeting to lysosome / protein targeting to vacuole / late endosome to vacuole transport / endosome organization / Dengue virus modulates apoptosis / RAB GEFs exchange GTP for GDP on RABs / pexophagy / positive regulation of natural killer cell mediated cytotoxicity / Golgi Cisternae Pericentriolar Stack Reorganization / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / vesicle transport along microtubule / COPII-mediated vesicle transport / cellular response to nitrogen starvation / negative regulation of programmed cell death / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / COPI-dependent Golgi-to-ER retrograde traffic / 1-phosphatidylinositol-3-kinase activity / post-transcriptional regulation of gene expression / virion assembly / response to vitamin E / transport vesicle membrane / Macroautophagy / endosome to lysosome transport / p38MAPK cascade / autophagosome membrane docking / response to iron(II) ion / Golgi organization / RSV-host interactions / cytoplasmic pattern recognition receptor signaling pathway / phosphatidylinositol phosphate biosynthetic process / negative regulation of protein phosphorylation / phosphatidylinositol-mediated signaling / autolysosome / autophagosome membrane / PI3K Cascade / autophagosome maturation / RHO GTPases Activate NADPH Oxidases / JNK cascade / mitotic metaphase chromosome alignment / endoplasmic reticulum to Golgi vesicle-mediated transport / axoneme / synaptic vesicle endocytosis / cellular response to glucose starvation / cellular defense response / autophagosome assembly / mitophagy / phosphatidylinositol 3-kinase binding / COPI-mediated anterograde transport / substrate adhesion-dependent cell spreading / regulation of macroautophagy / vesicle-mediated transport / positive regulation of intrinsic apoptotic signaling pathway / phagocytic vesicle / protein-membrane adaptor activity / endomembrane system / positive regulation of autophagy / response to endoplasmic reticulum stress / autophagosome / cellular response to epidermal growth factor stimulus / cellular response to copper ion
Similarity search - Function
Nuclear receptor-binding factor 2, C-terminal / Nuclear receptor-binding factor 2, MIT domain / Nuclear receptor-binding factor 2 / Nuclear receptor-binding factor 2, autophagy regulator / MIT domain of nuclear receptor-binding factor 2 / : / 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 ...Nuclear receptor-binding factor 2, C-terminal / Nuclear receptor-binding factor 2, MIT domain / Nuclear receptor-binding factor 2 / Nuclear receptor-binding factor 2, autophagy regulator / MIT domain of nuclear receptor-binding factor 2 / : / 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 / ARF-like small GTPases; ARF, ADP-ribosylation factor / 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. / 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-1A / Beclin-1 / Beclin 1-associated autophagy-related key regulator / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Nuclear receptor-binding factor 2 / Phosphoinositide 3-kinase regulatory subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 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
CitationJournal: To Be Published
Title: When VPS34 complexes double down: Two RAB5s for VPS34-CII, two RAB1s for NRBF2-dimerized VPS34-CI
Authors: Spokaite, S. / Ohashi, Y. / Dessus, A.N. / Bourguet, M. / Williams, R.L.
History
DepositionJul 10, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 22, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 22, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 22, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 22, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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: Beclin 1-associated autophagy-related key regulator
E: Ras-related protein Rab-1A
I: Beclin-1
L: Nuclear receptor-binding factor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)474,76814
Polymers473,3947
Non-polymers1,3747
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 , 6 types, 7 molecules ABCIDEL

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


Mass: 101680.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Full length untagged VPS34 / 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
Source method: isolated from a genetically manipulated source
Details: Full length VPS15 with 13 extra amino acids at the C-terminus left after TEV cleaving (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.: 2
Source method: isolated from a genetically manipulated source
Details: Full length untagged BECLIN1 / Source: (gene. exp.) Homo sapiens (human) / Gene: BECN1, GT197 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q14457
#4: Protein Beclin 1-associated autophagy-related key regulator / Barkor / Autophagy-related protein 14-like protein / Atg14L


Mass: 55461.348 Da / Num. of mol.: 1 / Mutation: N256S, insertion of T after M1
Source method: isolated from a genetically manipulated source
Details: Full length untagged ATG14L with a N256S mutation, and an insertion of T after M1
Source: (gene. exp.) Homo sapiens (human) / Gene: ATG14, ATG14L, KIAA0831 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q6ZNE5
#5: Protein Ras-related protein Rab-1A / YPT1-related protein


Mass: 22834.758 Da / Num. of mol.: 1 / Mutation: C26S, Q70L, C126S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAB1A, RAB1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 RIPL / References: UniProt: P62820, small monomeric GTPase
#6: Protein Nuclear receptor-binding factor 2 / NRBF-2 / Comodulator of PPAR and RXR


Mass: 34852.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Full length NRBF2 with an N-terminal His6 tag followed by a TEV cleavage site
Source: (gene. exp.) Homo sapiens (human) / Gene: NRBF2, COPR / Production host: Escherichia coli (E. coli) / Strain (production host): C41(DE3)OverExpress / References: UniProt: Q96F24

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Non-polymers , 5 types, 7 molecules

#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-MYR / MYRISTIC ACID


Mass: 228.371 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H28O2 / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#11: 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: Human VPS34-CI (VPS34/VPS15/BECLIN1/ATG14L) in complex with NRBF2 and RAB1A-GTP (C26S, C126S, Q70L)
Type: COMPLEX
Details: NRBF2 coiled coil domain density not observed in EM
Entity ID: #5-#6, #1, #4, #3 / Source: RECOMBINANT
Molecular weightValue: 0.419 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
2150 mMSodium chlorideNaCl1
31 mMTCEPC9H15O6P1
43 mMManganese chlorideMnCl21
51 mMAMPPNPC10H17N6O12P31
62 % w/wTrehaloseC12H26O131
70.01 % v/vNonidet P40 substitute(C2H4O)nC14H22O1
SpecimenConc.: 1.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was crosslinked with 20 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

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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: 2800 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: 2 / Num. of real images: 24736
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: 635719
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27517 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00424349
ELECTRON MICROSCOPYf_angle_d0.68332892
ELECTRON MICROSCOPYf_dihedral_angle_d7.1283236
ELECTRON MICROSCOPYf_chiral_restr0.0473644
ELECTRON MICROSCOPYf_plane_restr0.0064213

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