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- EMDB-63444: Zebrafish ovum lysosomal peptide:N-glycanase -

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Basic information

Entry
Database: EMDB / ID: EMD-63444
TitleZebrafish ovum lysosomal peptide:N-glycanase
Map dataPost-processed map (manual b-factor)
Sample
  • Complex: D. rerio Ngly2 dimer
    • Protein or peptide: Si:dkey-256h2.1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordspeptide:N-glycanase / PNGase / dimer / HYDROLASE
Function / homology
Function and homology information


oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced ascorbate as one donor, and incorporation of one atom of oxygen
Similarity search - Function
: / Peptide-N-glycosidase F, N-terminal / Peptide-N-glycosidase F, N terminal / Peptide-N-glycosidase F, C-terminal / Peptide-N-glycosidase F, N terminal / Peptide-N-glycosidase F, C terminal / PHM/PNGase F domain superfamily / Copper type II, ascorbate-dependent monooxygenase-like, C-terminal / PA domain superfamily
Similarity search - Domain/homology
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsHonda A / Kamada K / Burton-Smith RN / Murata K / Suzuki T
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22K14950 Japan
Japan Agency for Medical Research and Development (AMED)JP23/24gm1310003h0004/5 Japan
CitationJournal: J Biol Chem / Year: 2025
Title: Structural characterization of zebrafish Ngly2, an ovary-enriched acid PNGase required for egg-free glycan production.
Authors: Akinobu Honda / Katsuhiko Kamada / Junichi Seino / Hiroto Hirayama / Haruhiko Fujihira / Masashi Ueki / Toshiyuki Shiraki / Raymond N Burton-Smith / Kazuyoshi Murata / Nozomi Ishii / Ichiro ...Authors: Akinobu Honda / Katsuhiko Kamada / Junichi Seino / Hiroto Hirayama / Haruhiko Fujihira / Masashi Ueki / Toshiyuki Shiraki / Raymond N Burton-Smith / Kazuyoshi Murata / Nozomi Ishii / Ichiro Matsuo / Tadashi Suzuki /
Abstract: Peptide:N-glycanase (PNGase) is a deglycosylating enzyme acting on asparagine(N)-linked glycans on glycoproteins. It is well established that fish possesses two PNGases with distinct properties. One ...Peptide:N-glycanase (PNGase) is a deglycosylating enzyme acting on asparagine(N)-linked glycans on glycoproteins. It is well established that fish possesses two PNGases with distinct properties. One is a cytosolic PNGase (NGLY1 in humans), active at neutral pH and widely conserved among eukaryotes. The other is called acid PNGase and is found in fish embryos; it is active at acidic pH and is believed to be of lysosomal origin. The gene encoding the acid PNGase has not been identified in animals, and its evolutionary distribution has remained unknown. In this study, we identified the gene encoding the acid PNGase, which we named Ngly2, in zebrafish (Danio rerio). Interestingly, zebrafish Ngly2 was found to have structural similarity with bacterial PNGase (PNGase F) and indeed appeared to share common catalytic residues, despite the fact that these two enzymes exhibit quite distinct pH profiles. The structure of zebrafish Ngly2 was determined by cryo-EM, showing that it forms homodimers and that its substrate is accommodated in the cleft between the protease-associated domain and PNGase domain, where the catalytic residues are located. Tissue distribution analysis indicated that ngly2 was almost exclusively expressed in the ovary. A zebrafish ngly2-KO line was found to be fertile, survive well, and show no overt phenotypes, although it had significantly smaller fertilized eggs. It was also revealed that ngly2 KO resulted in a substantial reduction in the level of free oligosaccharides in fertilized eggs, implying that Ngly2, not Ngly1, is responsible for the formation of most, if not all, egg-free glycans.
History
DepositionFeb 14, 2025-
Header (metadata) releaseDec 24, 2025-
Map releaseDec 24, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_63444.png

Downloads & links

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Map

FileDownload / File: emd_63444.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map (manual b-factor)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 280 pix.
= 239.4 Å		0.86 Å/pix.
x 280 pix.
= 239.4 Å		0.86 Å/pix.
x 280 pix.
= 239.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0624
Minimum - Maximum-0.3585588 - 0.6229582
Average (Standard dev.)0.00048327926 (±0.015598458)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 239.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Post-processed map (CryoSPARC self-estimated b-factor)

Fileemd_63444_additional_1.map
AnnotationPost-processed map (CryoSPARC self-estimated b-factor)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_63444_half_map_1.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_63444_half_map_2.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : D. rerio Ngly2 dimer

EntireName: D. rerio Ngly2 dimer
Components
  • Complex: D. rerio Ngly2 dimer
    • Protein or peptide: Si:dkey-256h2.1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: D. rerio Ngly2 dimer

SupramoleculeName: D. rerio Ngly2 dimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)

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Macromolecule #1: Si:dkey-256h2.1

MacromoleculeName: Si:dkey-256h2.1 / type: protein_or_peptide / ID: 1 / Details: Ngly2 (PNGase) / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 77.054906 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AAPKDHLIHN HHKHEHAHAE HLGGSAWSHP QFEKGSGLEV LFQGPAEFAS SQGNDKAPKN LHLVKRNAIK TRPSDALGGL EPGDPAPAF QVHTLDGMFV YSPRNESGRA LIVHAFTNKS AFLECLWTWS ESLSDLLDYL PSSTEVLMLS MDETAEQDAL W MREQVYRA ...String:
AAPKDHLIHN HHKHEHAHAE HLGGSAWSHP QFEKGSGLEV LFQGPAEFAS SQGNDKAPKN LHLVKRNAIK TRPSDALGGL EPGDPAPAF QVHTLDGMFV YSPRNESGRA LIVHAFTNKS AFLECLWTWS ESLSDLLDYL PSSTEVLMLS MDETAEQDAL W MREQVYRA AAHRGKEILS RLHFSPTHVY NLGNWIPRVL YSWGCGGHNC GLGQVVFSSP DWKGPVIGKR LNARYDWLYA HW STDPYRL LDVGDGCAPV ASLKGAVAWV SEGGCSFFTK IKNMEKSNAT GVLVYALPGN NIQDMNCKGD ECFTSLHIPA SMV HFQPKV KEALQKGRPV NVKFQVTPSR SFFFGIDQRG VLSEMGWFLY PSFRFMAWQA QWFVFNDALL EQLSQPAVTV SVFD HHDMH GNAGAHAVVD LPADISPYDV LELDTSLSCP GRRDETCAHW DHTVQLFVCC NDSSPYCNQE LGRWVTAFRR GTGHW LTDV SPLIPLLNNK KCSFTMKTAP WAMPWMTTLN LRFSQSNKTE RLYPFEVMPL FNGGTFDKDY NRRYHEITFS IPAATK KVE LYAVITGHGS DDNNCGEFCV TSHYFLINRS INNTLVFEAA GSPLGCSLLV PKGGVPNECG TWLYGRGGWC DGLQVDP WR RDITSQLDMS GSNSVRYFGL FEGRDPNPKT DPGNILMYSY LVFYQ

UniProtKB: Si:dkey-256h2.1

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / 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: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Details: Self-generated ab initio model from raw particle stack
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 371054
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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