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- PDB-9mhg: Cryo EM reconstruction of PI3KC3-C1 in complex with Human RAB1A(Q... -

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Basic information

Entry
Database: PDB / ID: 9mhg
TitleCryo EM reconstruction of PI3KC3-C1 in complex with Human RAB1A(Q70L), VPS34 kinase domain in the inactive conformation
Components
  • Beclin 1-associated autophagy-related key regulator
  • Beclin-1
  • Phosphatidylinositol 3-kinase catalytic subunit type 3
  • Phosphoinositide 3-kinase regulatory subunit 4
  • Ras-related protein Rab-1A
KeywordsSIGNALING PROTEIN / Complex / GTPase / GTP-binding / Autophagy / Kinase / Lipid Kinase
Function / homology
Function and homology information


extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / regulation of triglyceride metabolic process / positive regulation of glycoprotein metabolic process / extrinsic component of phagophore assembly site membrane / nucleus-vacuole junction / cellular response to aluminum ion / positive regulation of protein lipidation / postsynaptic endosome / growth hormone secretion ...extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / regulation of triglyceride metabolic process / positive regulation of glycoprotein metabolic process / extrinsic component of phagophore assembly site membrane / nucleus-vacuole junction / cellular response to aluminum ion / positive regulation of protein lipidation / postsynaptic endosome / growth hormone secretion / Toll Like Receptor 9 (TLR9) Cascade / Synthesis of PIPs at the late endosome membrane / positive regulation of stress granule assembly / 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 / host-mediated activation of viral genome replication / presynaptic endosome / positive regulation of attachment of mitotic spindle microtubules to kinetochore / mitochondria-associated endoplasmic reticulum membrane contact site / COPII-coated vesicle cargo loading / negative regulation of lysosome organization / engulfment of apoptotic cell / Synthesis of PIPs at the Golgi membrane / phosphatidylinositol kinase activity / regulation of protein complex stability / positive regulation of autophagosome assembly / early endosome to late endosome transport / response to L-leucine / cytoplasmic side of mitochondrial outer membrane / negative regulation of autophagosome assembly / protein localization to phagophore assembly site / phosphatidylinositol 3-kinase regulator activity / receptor catabolic process / phagophore assembly site membrane / protein targeting to vacuole / RAB geranylgeranylation / protein targeting to lysosome / SMAD protein signal transduction / late endosome to vacuole transport / endosome organization / melanosome transport / pexophagy / RAB GEFs exchange GTP for GDP on RABs / Golgi Cisternae Pericentriolar Stack Reorganization / vesicle transport along microtubule / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / phosphatidylinositol-3-phosphate biosynthetic process / cellular response to nitrogen starvation / COPII-mediated vesicle transport / negative regulation of programmed cell death / phosphatidylinositol 3-kinase / COPI-dependent Golgi-to-ER retrograde traffic / 1-phosphatidylinositol-3-kinase activity / lysosome organization / response to vitamin E / post-transcriptional regulation of gene expression / response to iron(II) ion / mitotic metaphase chromosome alignment / cytoplasmic pattern recognition receptor signaling pathway / endosome to lysosome transport / transport vesicle membrane / virion assembly / positive regulation of cardiac muscle hypertrophy / phosphatidylinositol-mediated signaling / Macroautophagy / autophagosome membrane docking / RSV-host interactions / p38MAPK cascade / Golgi organization / phosphatidylinositol phosphate biosynthetic process / negative regulation of protein phosphorylation / autolysosome / autophagosome membrane / PI3K Cascade / axoneme / synaptic vesicle endocytosis / autophagosome assembly / RHO GTPases Activate NADPH Oxidases / autophagosome maturation / amyloid-beta metabolic process / regulation of macroautophagy / endoplasmic reticulum to Golgi vesicle-mediated transport / neuron development / cellular defense response / cellular response to glucose starvation / mitophagy / phosphatidylinositol 3-kinase binding / intercellular bridge / positive regulation of intrinsic apoptotic signaling pathway / COPI-mediated anterograde transport / vesicle-mediated transport / phagocytic vesicle / JNK cascade / protein-membrane adaptor activity / positive regulation of autophagy
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 / : / VPS15-like, helical domain / Beclin-1, BH3 domain / Beclin-1 BH3 domain, Bcl-2-interacting / Atg6/Beclin / Atg6/Beclin C-terminal domain superfamily / Atg6, BARA domain ...UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / : / VPS15-like, helical domain / 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 / ARF-like small GTPases; ARF, ADP-ribosylation factor / 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 / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic 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
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 / Phosphoinositide 3-kinase regulatory subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCook, A.S.I. / Hurley, J.H. / Chen, M.
Funding support United States, 1items
OrganizationGrant numberCountry
Aligning Science Across Parkinsons (ASAP)ASAP-000350 United States
CitationJournal: Science / Year: 2025
Title: Structural pathway for PI3-kinase regulation by VPS15 in autophagy.
Authors: Annan S I Cook / Minghao Chen / Thanh N Nguyen / Ainara Claveras Cabezudo / Grace Khuu / Shanlin Rao / Samantha N Garcia / Mingxuan Yang / Anthony T Iavarone / Xuefeng Ren / Michael Lazarou ...Authors: Annan S I Cook / Minghao Chen / Thanh N Nguyen / Ainara Claveras Cabezudo / Grace Khuu / Shanlin Rao / Samantha N Garcia / Mingxuan Yang / Anthony T Iavarone / Xuefeng Ren / Michael Lazarou / Gerhard Hummer / James H Hurley /
Abstract: The class III phosphatidylinositol-3 kinase complexes I and II (PI3KC3-C1 and PI3KC3-C2) have vital roles in macroautophagy and endosomal maturation, respectively. We elucidated a structural pathway ...The class III phosphatidylinositol-3 kinase complexes I and II (PI3KC3-C1 and PI3KC3-C2) have vital roles in macroautophagy and endosomal maturation, respectively. We elucidated a structural pathway of enzyme activation through cryo-electron microscopy analysis of PI3KC3-C1. The inactive conformation of the VPS15 pseudokinase stabilizes the inactive conformation, sequestering its -myristate in the N-lobe of the pseudokinase. Upon activation, the myristate is liberated such that the VPS34 lipid kinase catalyzes phosphatidylinositol-3 phosphate production on membranes. The VPS15 pseudokinase domain binds tightly to guanosine triphosphate and stabilizes a web of interactions to autoinhibit the cytosolic complex and promote activation upon membrane binding. These findings show in atomistic detail how the VPS34 lipid kinase is activated in the context of a complete PI3K complex.
History
DepositionDec 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 3, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphoinositide 3-kinase regulatory subunit 4
B: Phosphatidylinositol 3-kinase catalytic subunit type 3
C: Beclin 1-associated autophagy-related key regulator
D: Beclin-1
E: Ras-related protein Rab-1A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)394,35910
Polymers393,0365
Non-polymers1,3235
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 5 molecules ABCDE

#1: Protein Phosphoinositide 3-kinase regulatory subunit 4 / PI3-kinase regulatory subunit 4 / PI3-kinase p150 subunit / Phosphoinositide 3-kinase adaptor protein


Mass: 158808.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3R4, VPS15 / Cell line (production host): HEK293T / Production host: Homo sapiens (human)
References: UniProt: Q99570, non-specific serine/threonine protein kinase
#2: 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: 101680.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3C3, VPS34 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q8NEB9, phosphatidylinositol 3-kinase
#3: Protein Beclin 1-associated autophagy-related key regulator


Mass: 55387.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATG14 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q6ZNE5
#4: 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): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q14457
#5: Protein Ras-related protein Rab-1A / YPT1-related protein


Mass: 25206.527 Da / Num. of mol.: 1 / Mutation: Q70L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAB1A, RAB1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P62820, small monomeric GTPase

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Non-polymers , 3 types, 5 molecules

#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-MYR / MYRISTIC ACID


Mass: 228.371 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H28O2 / 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

ComponentName: Phosphatidylinositol-3 kinase class III complex I bound to RAB1A
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.384450 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4 / Details: OG supplemented at time of grid preparation 0.05%
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2200 mMsodium chlorideNaCl1
32 mMMagnesium chlorideMgCl21
41 mMTCEPC9H15O6P1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse sample
Specimen supportDetails: 25 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil Active R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: 100% humidity, 3 s wait time, blot force -15

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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: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 19300
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2Topazparticle selection
8ISOLDE1.7model fitting
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
14PHENIX1.21.2_5419model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161381 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 95.68 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006323096
ELECTRON MICROSCOPYf_angle_d0.877131220
ELECTRON MICROSCOPYf_chiral_restr0.05213454
ELECTRON MICROSCOPYf_plane_restr0.00874003
ELECTRON MICROSCOPYf_dihedral_angle_d14.748749

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