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- EMDB-48258: PI3KC3C1 bound to RAB1A, Inactive state, VPS34 kinase domain loca... -

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Basic information

Entry
Database: EMDB / ID: EMD-48258
TitlePI3KC3C1 bound to RAB1A, Inactive state, VPS34 kinase domain local refinement
Map data
Sample
  • Complex: Phosphatidylinositol-3 kinase class III complex I bound to RAB1A
KeywordsComplex / GTPase / GTP-binding / Autophagy / Kinase / Lipid Kinase / SIGNALING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCook ASI / Chen M / Hurley JH
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateSep 3, 2025-
Current statusSep 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48258.png

Downloads & links

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Map

FileDownload / File: emd_48258.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 400 pix.
= 419.2 Å		1.05 Å/pix.
x 400 pix.
= 419.2 Å		1.05 Å/pix.
x 400 pix.
= 419.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.225
Minimum - Maximum-1.258316 - 1.8528455
Average (Standard dev.)-0.00037069718 (±0.030408822)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48258_msk_1.map
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Half map: #1

Fileemd_48258_half_map_1.map
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Half map: #2

Fileemd_48258_half_map_2.map
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Sample components

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Entire : Phosphatidylinositol-3 kinase class III complex I bound to RAB1A

EntireName: Phosphatidylinositol-3 kinase class III complex I bound to RAB1A
Components
  • Complex: Phosphatidylinositol-3 kinase class III complex I bound to RAB1A

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Supramolecule #1: Phosphatidylinositol-3 kinase class III complex I bound to RAB1A

SupramoleculeName: Phosphatidylinositol-3 kinase class III complex I bound to RAB1A
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 384.45 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
200.0 mMNaClsodium chloride
2.0 mMMgCl2Magnesium chloride
1.0 mMC9H15O6PTCEP

Details: OG supplemented at time of grid preparation 0.05%
GridModel: Quantifoil Active R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailsmonodisperse sample

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 19300 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.1) / Type: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 161381
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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