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- EMDB-66011: Structural Analysis of a Plant Glycoside Hydrolase Family 116 Glu... -

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Basic information

Entry
Database: EMDB / ID: EMD-66011
TitleStructural Analysis of a Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase by Cryogenic Electron Microscopy (Cryo-EM)
Map data
Sample
  • Complex: Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase Hexameric Complex
    • Protein or peptide: Non-lysosomal glucosylceramidase
KeywordsGlucosylceramide / AtGCD3 / plant / GH116 / GBA2 / glucosylceramidase / HYDROLASE
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)
Methodsingle 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 JR
Funding support Thailand, 1 items
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, 2025-
Header (metadata) releaseApr 29, 2026-
Map releaseApr 29, 2026-
UpdateApr 29, 2026-
Current statusApr 29, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_66011.png

Downloads & links

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Map

FileDownload / File: emd_66011.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 416 pix.
= 395.2 Å		0.95 Å/pix.
x 416 pix.
= 395.2 Å		0.95 Å/pix.
x 416 pix.
= 395.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.28
Minimum - Maximum-0.1375462 - 1.4600177
Average (Standard dev.)0.0034370138 (±0.05261781)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 395.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_66011_msk_1.map
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Half map: #2

Fileemd_66011_half_map_1.map
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Half map: #1

Fileemd_66011_half_map_2.map
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Sample components

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Entire : Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase Hexameri...

EntireName: Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase Hexameric Complex
Components
  • Complex: Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase Hexameric Complex
    • Protein or peptide: Non-lysosomal glucosylceramidase

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Supramolecule #1: Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase Hexameri...

SupramoleculeName: Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase Hexameric Complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Non-lysosomal glucosylceramidase

MacromoleculeName: Non-lysosomal glucosylceramidase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: glucosylceramidase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 104.640656 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GGGMEKNGHT ESELQTQMVG NERLPQVTWQ RKLNSKVKNP SEFKMSIRDV LHLFPLGYRL WRYTKEEAKK GRFSMYDIFK KRHVRGDHG VPLGGIGGGS IGRSYKGEFQ QFKLFPKICE EAPILTNQFS VFVSRPGGLS YSTVLCPTKP KSVKGKTEDL G IESWDWNM ...String:
GGGMEKNGHT ESELQTQMVG NERLPQVTWQ RKLNSKVKNP SEFKMSIRDV LHLFPLGYRL WRYTKEEAKK GRFSMYDIFK KRHVRGDHG VPLGGIGGGS IGRSYKGEFQ QFKLFPKICE EAPILTNQFS VFVSRPGGLS YSTVLCPTKP KSVKGKTEDL G IESWDWNM EGDKSTYHAL YPRSWTVYNE PDPELRIVSR QVSPFIPHNY KESSLPVSVF EFTMSNLGKE EATVTLLFTW EN SVGGASG LTGEHFNSTI MERDGVHAIV LHHKTGNGHP PVTYAIAAQE TEDVHVSECP CFLVSGHSPK EITAKEMWDE IKK NKSFDE LNSEPGSPSR PGTSIGAAIA AKVKVPPGCD RTVTFSLSWD CPEVRFNEKT YHRRYTKFYG NLGDAAVKMA RDAL LNYVD WESQIEAWQS PILSDTTLPD WYRVTLFNEL YYFNSGGTIW TDGLPPKESI ERSKVTNTEQ NDIVIDLFQK INAVC EQIY SPQSSNSEEN IGQFIYLEGI EYLMYNTYDV HFYSSFALLS LFPKLALSIQ RDFAATVLIQ DPTKKKIMSS GEWVTR KLL GSVPHDIGLN DPWLELNEYN FFNTDRWKDL NAKFVLQVYR DVVATNDQSF AKAVWPSVYT AVAYLDQFDK DEDGMIE NE GFPDQTYDAW SVTGVSAYCG GLWVAALQAA SAFASIVGEN AVAIYFNAKY EKAKIVYEKL WNGSYFNYDD SGSGSSSS I LADQLAGQWY ARACGLKPIT KEEWIKKALE TIYEFNVMKV KGGTRGAVNG MSTEGKVDTN SLVSKEVWAG TTYSVAACM IQEGQREKGF QTASGIYEAI WSDRGLSCSF QTPEAWNMND EYRSLCYMRP LAIWAIQWAL TRTQSFGEEK QKLVAGDEEE ESNLLLRQH KGFKDVARFV KIVPTSNVHR SRLQHTYETV LKTLRL

UniProtKB: Non-lysosomal glucosylceramidase

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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

Concentration0.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNa2HPO4sodium phosphate
150.0 mMKClpotassium chloride
1.0 mMC4H10O2S2dithiothreitol
1.0 mM[CH2N(CH2CO2H)2]2EDTA
10.0 %C3O3H8glycerol

Details: 50mM potassium phosphate, 150mM KCl, 1mM DTT, 1mM EDTA, 10% glycerol
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 1200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Chamber temperature: 274 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 38.64 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.9 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 150000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 210468
CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold 2 model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 33669
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC (ver. 4.7.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
SoftwareName: UCSF ChimeraX (ver. 1.10)
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9wiz:
Structural Analysis of a Plant Glycoside Hydrolase Family 116 Glucosyl Ceramidase by Cryogenic Electron Microscopy (Cryo-EM)

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