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- EMDB-54320: VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RA... -

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Basic information

Entry
Database: EMDB / ID: EMD-54320
TitleVPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RAB5A (Q79L) on the VPS15 subunit, focused refinement of the RAB5A interface with VPS15
Map data
Sample
  • Complex: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
KeywordsLipid kinase / GTPase / kinase / endocytic sorting / endosome maturation / SIGNALING PROTEIN / ENDOCYTOSIS
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsSpokaite S / Ohashi Y / Bourguet M / Dessus 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
Citation
Journal: Elife / Year: 2026
Title: A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution.
Authors: Saule Spokaite / Yohei Ohashi / Maxime Bourguet / Antoine Nicolas Dessus / Roger L Williams /
Abstract: RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by ...RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in VPS15 resulted in defective CPY sorting, loss of colocalisation with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.
#1: Journal: Elife / Year: 2026
Title: A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution
Authors: Spokaite S / Ohashi Y / Bourguet M / Dessus AN / Williams RL
History
DepositionJul 9, 2025-
Header (metadata) releaseJun 3, 2026-
Map releaseJun 3, 2026-
UpdateJun 10, 2026-
Current statusJun 10, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54320.png

Downloads & links

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Map

FileDownload / File: emd_54320.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.38132274 - 1.0237669
Average (Standard dev.)0.002891342 (±0.022781078)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 365.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_54320_msk_1.map
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Half map: #1

Fileemd_54320_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #2

Fileemd_54320_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/...

EntireName: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
Components
  • Complex: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit

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Supramolecule #1: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/...

SupramoleculeName: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 409 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

Concentration2.45 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium chloride
1.0 mMC9H15O6PTCEP
5.0 mMMgCl2Magnesium chloride
1.0 mMC10H17N6O12P3AMPPNP
4.0 mMC32H58N2O8SCHAPSO
0.005 % v/v(C2H4O)nC14H22ONonidet P40 substitute
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 287 K / Instrument: FEI VITROBOT MARK III
Details: 3.5 uL sample, 0 second wait, 3.5 second blot, blot force +8.
DetailsSample was crosslinked with 30 uM BS3 for 20 minutes on ice, then quenched with 100 mM TRIS pH 8.0 prior to vitrification

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
SoftwareName: EPU
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 13301 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 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

Particle selectionNumber selected: 370889
CTF correctionType: NONE
Startup modelType of model: OTHER
Details: Density from VPS34-CI bound to NRBF2 MIT domain, determined earlier
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 84296
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: ISOLDE
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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