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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 / growth hormone secretion / Toll Like Receptor 9 (TLR9) Cascade / postsynaptic endosome ...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 / growth hormone secretion / Toll Like Receptor 9 (TLR9) Cascade / postsynaptic endosome / Synthesis of PIPs at the late endosome membrane / phosphatidylinositol 3-kinase complex, class III / engulfment of apoptotic cell / 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 / presynaptic endosome / positive regulation of stress granule assembly / response to mitochondrial depolarisation / positive regulation of protein lipidation / positive regulation by host of viral genome replication / positive regulation of attachment of mitotic spindle microtubules to kinetochore / COPII-coated vesicle cargo loading / mitochondria-associated endoplasmic reticulum membrane contact site / melanosome transport / negative regulation of lysosome organization / Synthesis of PIPs at the Golgi membrane / phosphatidylinositol kinase activity / regulation of protein complex stability / positive regulation of autophagosome assembly / phosphatidylinositol 3-kinase regulator activity / negative regulation of autophagosome assembly / cytoplasmic side of mitochondrial outer membrane / protein localization to phagophore assembly site / vesicle transport along microtubule / receptor catabolic process / phagophore assembly site membrane / protein targeting to vacuole / RAB geranylgeranylation / SMAD protein signal transduction / protein targeting to lysosome / early endosome to late endosome transport / late endosome to vacuole transport / pexophagy / RAB GEFs exchange GTP for GDP on RABs / Golgi Cisternae Pericentriolar Stack Reorganization / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / phosphatidylinositol-3-phosphate biosynthetic process / COPII-mediated vesicle transport / cellular response to nitrogen starvation / negative regulation of programmed cell death / response to iron(II) ion / phosphatidylinositol 3-kinase / lysosome organization / COPI-dependent Golgi-to-ER retrograde traffic / 1-phosphatidylinositol-3-kinase activity / mitotic metaphase chromosome alignment / post-transcriptional regulation of gene expression / cytoplasmic pattern recognition receptor signaling pathway / autophagosome membrane docking / transport vesicle membrane / endosome to lysosome transport / Macroautophagy / virion assembly / phosphatidylinositol-mediated signaling / Golgi organization / positive regulation of cardiac muscle hypertrophy / RSV-host interactions / p38MAPK cascade / phosphatidylinositol phosphate biosynthetic process / autolysosome / regulation of protein phosphorylation / autophagosome membrane / negative regulation of protein phosphorylation / synaptic vesicle endocytosis / PI3K Cascade / axoneme / autophagosome assembly / RHO GTPases Activate NADPH Oxidases / response to vitamin E / autophagosome maturation / amyloid-beta metabolic process / regulation of macroautophagy / endoplasmic reticulum to Golgi vesicle-mediated transport / neuron development / phosphatidylinositol 3-kinase binding / cellular defense response / cellular response to glucose starvation / mitophagy / COPI-mediated anterograde transport / positive regulation of intrinsic apoptotic signaling pathway / protein-membrane adaptor activity / vesicle-mediated transport / phagocytic vesicle / JNK cascade / cellular response to copper ion / positive regulation of autophagy / endomembrane system
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 Rab1 family 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 / Armadillo-type fold / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / 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: To Be Published
Title: Structural pathway for class III PI 3-kinase activation by the myristoylated GTPbinding pseudokinase VPS15
Authors: Cook, A.S.I. / Chen, M. / Ngyuen, T.N. / Claveras-Cabezudo, A. / Khuu, G. / Rao, S. / Garcia, S.N. / Yang, M. / Iavarone, A.T. / Ren, X. / Lazarou, M. / Hummer, G. / Hurley, J.H.
History
DepositionDec 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release

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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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