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- PDB-9wiz: Structural Analysis of a Plant Glycoside Hydrolase Family 116 Glu... -

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Entry
Database: PDB / ID: 9wiz
TitleStructural Analysis of a Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase by Cryogenic Electron Microscopy (Cryo-EM)
ComponentsNon-lysosomal glucosylceramidase
KeywordsHYDROLASE / Glucosylceramide / AtGCD3 / plant / GH116 / GBA2 / glucosylceramidase
Function / homology
Function and homology information


glucosylceramidase / glucosylceramide catabolic process / glucosylceramidase activity / sphingolipid metabolic process / plant-type vacuole / beta-glucosidase activity / carbohydrate metabolic process / endoplasmic reticulum / plasma membrane
Similarity search - Function
Glycosyl-hydrolase family 116, catalytic region / Beta-glucosidase GBA2-type / Glycosyl-hydrolase family 116, N-terminal / : / Glycosyl-hydrolase family 116, catalytic region / beta-glucosidase 2, glycosyl-hydrolase family 116 N-term / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily
Similarity search - Domain/homology
Non-lysosomal glucosylceramidase
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsChoknud, S. / Rungsarityotin, W. / Ounjai, P. / Mo-mai, P. / Wangkanont, K. / Arthanareeswaran, K. / Beagbandee, C. / Ketudat-Cairns, J.R.
Funding support Thailand, 1items
OrganizationGrant numberCountry
The Thailand Research Fund (TRF) Thailand
CitationJournal: Int J Biol Macromol / Year: 2026
Title: Structural analysis of the plant glycoside hydrolase family 116 glucosylceramidase AtGCD3 by cryogenic electron microscopy.
Authors: Sunaree Choknud / Karunambigai Arthanareeswaran / Wasinee Rungsarityotin / Chamaipon Beagbandee / Surapoj Sanram / Nuntaporn Kamonsutthipaijit / Pornphawit Mo-Mai / Rung-Yi Lai / Wipa ...Authors: Sunaree Choknud / Karunambigai Arthanareeswaran / Wasinee Rungsarityotin / Chamaipon Beagbandee / Surapoj Sanram / Nuntaporn Kamonsutthipaijit / Pornphawit Mo-Mai / Rung-Yi Lai / Wipa Suginta / Kittikhun Wangkanont / Puey Ounjai / James R Ketudat Cairns /
Abstract: Glucosylceramide (GlcCer) is a common glycosphingolipid that accumulates in cells in response to Gaucher disease, diabetes, and skin disorders in humans and is also found in plants. In animals, ...Glucosylceramide (GlcCer) is a common glycosphingolipid that accumulates in cells in response to Gaucher disease, diabetes, and skin disorders in humans and is also found in plants. In animals, GlcCer is catabolized by glucosylcerebrosidase 1 and 2 (GBA1 and GBA2). GBA1 is a lysosomal enzyme in glycoside hydrolase (GH) family GH30, while GBA2 is a membrane-associated cytoplasmic protein in family GH116. Currently, there are no experimental structures of eukaryotic GH116 homologues. Although the bacterial TxGH116 β-glucosidase structure was determined by X-ray crystallography, TxGH116 does not hydrolyze glucosylceramides, unlike the animal and plant enzymes. Therefore, we have investigated the structure of plant GH116 (AtGCD3) by cryogenic electron microscopy (Cryo-EM) single-particle analysis. The recombinant AtGCD3 protein was produced in Escherichia coli and purified by immobilized-metal affinity chromatography followed by size-exclusion chromatography. The Cryo-EM structure revealed a unique hexameric arrangement, composed of a dimer of trimers. Hydrophobic interactions and hydrogen bonds stabilize each trimer at the trimer interface. The two trimers stack face-to-face with a slight twist, with salt bridges and hydrogen bonding at their interface. Two α-helices not found in previously described GH116 structures cover the active site, forming two hydrophobic channels that may be involved in glucosylceramide binding. Molecular dynamics simulations showed that glucosylceramide can bind stably in the active site with its lipid tails in these channels. This first eukaryotic structure of a GH116 enzyme generates a template for improved modeling of human GBA2, with implications for treating human diseases, such as Gaucher disease and hereditary spastic paraplegia.
History
DepositionAug 30, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 29, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Apr 29, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Non-lysosomal glucosylceramidase
B: Non-lysosomal glucosylceramidase
C: Non-lysosomal glucosylceramidase
D: Non-lysosomal glucosylceramidase
F: Non-lysosomal glucosylceramidase
E: Non-lysosomal glucosylceramidase


Theoretical massNumber of molelcules
Total (without water)627,8446
Polymers627,8446
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Non-lysosomal glucosylceramidase / NLGase


Mass: 104640.656 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: GCD3, At4g10060, F28M11.3 / Plasmid: pET30a(+) / Details (production host): pET30/At4GH116 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: F4JLJ2, glucosylceramidase
Has protein modificationN

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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: Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase Hexameric Complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Details: 50mM potassium phosphate, 150mM KCl, 1mM DTT, 1mM EDTA, 10% glycerol
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium phosphateNa2HPO41
2150 mMpotassium chlorideKCl1
31 mMdithiothreitolC4H10O2S21
41 mMEDTA[CH2N(CH2CO2H)2]21
510 %glycerolC3O3H81
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Chamber temperature: 274 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 3900 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 38.64 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.1particle selection
4cryoSPARC4.7.1CTF correction
7UCSF ChimeraX1.1model fitting
9PHENIX1.21.2.5419model refinement
10cryoSPARC4.7.1initial Euler assignment
11cryoSPARC4.7.1final Euler assignment
12cryoSPARC4.7.1classification
13cryoSPARC4.7.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 210468
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33669 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE

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