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- PDB-9tw2: Cryo-EM structure of human VPS34-CI in complex with GABARAP -

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Basic information

Entry
Database: PDB / ID: 9tw2
TitleCryo-EM structure of human VPS34-CI in complex with GABARAP
Components
  • Beclin 1-associated autophagy-related key regulator
  • Beclin-1
  • Gamma-aminobutyric acid receptor-associated protein
  • Nuclear receptor-binding factor 2
  • Phosphatidylinositol 3-kinase catalytic subunit type 3
  • Phosphoinositide 3-kinase regulatory subunit 4
KeywordsLIPID BINDING PROTEIN / Lipid kinase / Complex / Autophagy
Function / homology
Function and homology information


extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / extrinsic component of phagophore assembly site membrane / 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 / positive regulation of protein K48-linked ubiquitination ...extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / extrinsic component of phagophore assembly site membrane / 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 / positive regulation of protein K48-linked ubiquitination / 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 / regulation of Rac protein signal transduction / positive regulation of attachment of mitotic spindle microtubules to kinetochore / host-mediated activation of viral genome replication / engulfment of apoptotic cell / regulation of protein complex stability / negative regulation of lysosome organization / mitochondria-associated endoplasmic reticulum membrane contact site / 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 / response to L-leucine / protein targeting to lysosome / protein targeting to vacuole / late endosome to vacuole transport / endosome organization / GABA receptor binding / pexophagy / Dengue virus modulates apoptosis / phosphatidylethanolamine binding / 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 / microtubule associated complex / negative regulation of programmed cell death / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / post-transcriptional regulation of gene expression / response to vitamin E / extrinsic apoptotic signaling pathway via death domain receptors / Macroautophagy / endosome to lysosome transport / p38MAPK cascade / autophagosome membrane docking / response to iron(II) ion / RSV-host interactions / cytoplasmic pattern recognition receptor signaling pathway / phosphatidylinositol phosphate biosynthetic process / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / negative regulation of protein phosphorylation / smooth endoplasmic reticulum / phosphatidylinositol-mediated signaling / autolysosome / autophagosome membrane / PI3K Cascade / autophagosome maturation / protein targeting / JNK cascade / RHO GTPases Activate NADPH Oxidases / mitotic metaphase chromosome alignment / axoneme / cellular response to glucose starvation / synaptic vesicle endocytosis / cellular defense response / autophagosome assembly / mitophagy / phosphatidylinositol 3-kinase binding / beta-tubulin binding / regulation of macroautophagy / positive regulation of intrinsic apoptotic signaling pathway / phagocytic vesicle / protein-membrane adaptor activity / positive regulation of autophagy / response to endoplasmic reticulum stress / autophagosome / cellular response to epidermal growth factor stimulus / cellular response to copper ion / cellular response to amino acid starvation / cellular response to starvation / regulation of autophagy / macroautophagy / regulation of cytokinesis / phosphatidylinositol 3-kinase/protein kinase B signal transduction / Antigen Presentation: Folding, assembly and peptide loading of class I MHC
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 / Beclin-1 BH3 domain, Bcl-2-interacting ...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 / Autophagy protein Atg8 ubiquitin-like / Autophagy protein Atg8 ubiquitin like / HEAT, type 2 / HEAT repeat 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 / C2 domain superfamily / Armadillo-like helical / WD domain, G-beta repeat / Armadillo-type fold / Ubiquitin-like domain superfamily / 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
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / MYRISTIC ACID / Gamma-aminobutyric acid receptor-associated protein / 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: 3.58 Å
AuthorsDessus, A.N. / 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: Cryo-EM structure of human VPS34-CI in complex with GABARAP
Authors: Dessus, A.N. / Williams, R.L.
History
DepositionJan 13, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 5, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 5, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 5, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 5, 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

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: Beclin 1-associated autophagy-related key regulator
E: Nuclear receptor-binding factor 2
F: Gamma-aminobutyric acid receptor-associated protein
G: Beclin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)440,49212
Polymers439,6667
Non-polymers8275
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 ABCGDEF

#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
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3C3, VPS34 / 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: 154790.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal tag (TEV cut). / Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3R4, VPS15 / 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
Source: (gene. exp.) Homo sapiens (human) / Gene: BECN1, GT197 / 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
Source method: isolated from a genetically manipulated source
Details: Insertion of a Threonine at position 2 / Source: (gene. exp.) Homo sapiens (human) / Gene: ATG14, ATG14L, KIAA0831 / Production host: Homo sapiens (human) / References: UniProt: Q6ZNE5
#5: Protein Nuclear receptor-binding factor 2 / NRBF-2 / Comodulator of PPAR and RXR


Mass: 9716.190 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MIT domain (1-84) / Source: (gene. exp.) Homo sapiens (human) / Gene: NRBF2, COPR / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q96F24
#6: Protein Gamma-aminobutyric acid receptor-associated protein / GABA(A) receptor-associated protein / MM46


Mass: 14111.165 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal tag + deletion of L117 / Source: (gene. exp.) Homo sapiens (human) / Gene: GABARAP, FLC3B, HT004 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O95166

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Non-polymers , 4 types, 5 molecules

#7: Chemical ChemComp-MYR / MYRISTIC ACID


Mass: 228.371 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H28O2 / Feature type: SUBJECT OF INVESTIGATION
#8: 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
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / 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

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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

Component
IDNameTypeEntity IDParent-IDSource
1Protein complex of VPS34-CI, NRBF2 MIT and GABARAPCOMPLEX#1-#60RECOMBINANT
2Human VPS34-CICOMPLEX#1-#41RECOMBINANT
3NRBF2 MIT domainCOMPLEX#51RECOMBINANT
4GABARAPCOMPLEX#61RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.387212 MDaNO
210.363427 MDaNO
310.09699 MDaNO
410.14086 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
21Homo sapiens (human)9606
32Homo sapiens (human)9606pCAG
43Escherichia coli BL21(DE3) (bacteria)469008
54Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESHEPES1
2100 mMsodium chlorideNaCl1
31 mMTris-(2-Carboxyethyl)phosphineTCEP1
44 mMCHAPSOCHAPSO1
50.005 % (v/v)Nonidet P-40Nonidet-P-401
SpecimenConc.: 1.92 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 3.6 uM VPS34-CI was mixed with 3.6 uM NRBF2 MIT and 40 uM GABARAP.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 287.15 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: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.97 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 18226
Image scansWidth: 4092 / Height: 5760

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Processing

EM software
IDNameVersionCategoryImage processing-ID
1crYOLO1.7.5particle selection1
2EPUimage acquisition
4cryoSPARC4.6.2CTF correction1
7UCSF ChimeraX1.7.1model fitting
8ISOLDE1.7.1model fitting
10TEMPy1.2.0model refinement
11PHENIX1.21.2_5419model refinement
12cryoSPARC4.6.2initial Euler assignment1
13cryoSPARC4.6.2final Euler assignment1
14cryoSPARC4.6.2classification1
15cryoSPARC4.6.23D reconstruction1
16crYOLO1.7.5particle selection2
17cryoSPARC4.6.2CTF correction2
18cryoSPARC4.6.2initial Euler assignment2
19cryoSPARC4.6.2final Euler assignment2
20cryoSPARC4.6.2classification2
21cryoSPARC4.6.23D reconstruction2
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing-IDNum. of particles selected
11973900
22973900
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
13.58FSC 0.5 CUT-OFF55368819TW2POINT
23.58FSC 0.5 CUT-OFF55368819TW2POINT
33.58FSC 0.5 CUT-OFF55368829TW2POINT
43.58FSC 0.5 CUT-OFF55368829TW2POINT
Atomic model buildingSpace: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.58 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00423601
ELECTRON MICROSCOPYf_angle_d0.68731874
ELECTRON MICROSCOPYf_dihedral_angle_d6.6293133
ELECTRON MICROSCOPYf_chiral_restr0.0473522
ELECTRON MICROSCOPYf_plane_restr0.0064089

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