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- EMDB-56345: Cryo-EM structure of human VPS34-CI in complex with GABARAP - alt... -

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Basic information

Entry
Database: EMDB / ID: EMD-56345
TitleCryo-EM structure of human VPS34-CI in complex with GABARAP - alternative conformation, consensus map
Map dataConsensus map for alternative conformation
Sample
  • Complex: Protein complex of VPS34-CI, NRBF2 MIT and GABARAP
    • Complex: Human VPS34-CI
    • Complex: NRBF2 MIT domain
    • Complex: GABARAP
KeywordsLipid kinase / Complex / Autophagy / LIPID BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDessus AN / Williams RL
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105184308 United Kingdom
Cancer Research UKDRCPGM 100014 United Kingdom
CitationJournal: Nat Commun / Year: 2026
Title: A GABARAP-PtdIns3K-C1 positive feedback loop at the heart of the phagophore nucleation.
Authors: Antoine N Dessus / Yohei Ohashi / Maxime Bourguet / Tomos E Morgan / Anastasia Nunez / Maria Manifava / Nicholas T Ktistakis / Roger L Williams /
Abstract: In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized remains elusive. ...In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized remains elusive. The phagophore initiation needs the lipid kinase PIK3C3-Complex 1 (PtdIns3K-C1), which produces phosphatidylinositol-3-phosphate (PtdIns3P). PtdIns3P recruits WIPI2 that facilitates lipidation of mammalian ATG8 (mATG8) family proteins on phagophores. Here we show that recombinant membrane-coupled GABARAP binds to and potently activates PtdIns3K-C1. By a combination of cryo-electron microscopy, structural mass spectrometry, activity assays and mutagenesis, we show that GABARAP activates PtdIns3K-C1 through two binding sites. We propose that once GABARAP is indirectly recruited by PtdIns3P generated by basal activity of PtdIns3K-C1, a positive feedback loop is formed where PtdIns3K-C1 interacts with GABARAP and becomes activated to produce more PtdIns3P, thereby further stimulating GABARAP lipidation. This mechanism would be central for autophagosome biogenesis, where enlarged membranes need to be rapidly synthesized.
History
DepositionJan 13, 2026-
Header (metadata) releaseAug 5, 2026-
Map releaseAug 5, 2026-
UpdateSep 16, 2026-
Current statusSep 16, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56345.png

Downloads & links

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Map

FileDownload / File: emd_56345.map.gz / Format: CCP4 / Size: 352.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus map for alternative conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 452 pix.
= 327.7 Å		0.73 Å/pix.
x 452 pix.
= 327.7 Å		0.73 Å/pix.
x 452 pix.
= 327.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.725 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0691
Minimum - Maximum-0.09217343 - 0.22950025
Average (Standard dev.)0.00039992898 (±0.011342171)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions452452452
Spacing452452452
CellA=B=C: 327.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_56345_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_56345_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Protein complex of VPS34-CI, NRBF2 MIT and GABARAP

EntireName: Protein complex of VPS34-CI, NRBF2 MIT and GABARAP
Components
  • Complex: Protein complex of VPS34-CI, NRBF2 MIT and GABARAP
    • Complex: Human VPS34-CI
    • Complex: NRBF2 MIT domain
    • Complex: GABARAP

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Supramolecule #1: Protein complex of VPS34-CI, NRBF2 MIT and GABARAP

SupramoleculeName: Protein complex of VPS34-CI, NRBF2 MIT and GABARAP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 140.86 KDa

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Supramolecule #2: Human VPS34-CI

SupramoleculeName: Human VPS34-CI / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: NRBF2 MIT domain

SupramoleculeName: NRBF2 MIT domain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: GABARAP

SupramoleculeName: GABARAP / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Homo sapiens (human)

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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

Concentration1.92 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
100.0 mMNaClsodium chloride
1.0 mMTCEPTris-(2-Carboxyethyl)phosphine
4.0 mMCHAPSOCHAPSO
0.005 % (v/v)Nonidet-P-40Nonidet P-40
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287.15 K / Instrument: FEI VITROBOT MARK II
Details3.6 uM VPS34-CI was mixed with 3.6 uM NRBF2 MIT and 40 uM GABARAP.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number grids imaged: 2 / Number real images: 18226 / Average exposure time: 0.97 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination 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: 105000
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 #1

Image processing ID1
Particle selectionNumber selected: 973900
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

54326

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 577838
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.6.2)

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Image processing #2

Image processing ID2
Particle selectionNumber selected: 973900
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

54326

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 577838
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.6.2)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Overall B value: 81.56

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