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- EMDB-30910: Structure of Drosophila melanogaster GlcNAc-1-phosphotransferase -

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Basic information

Entry
Database: EMDB / ID: EMD-30910
TitleStructure of Drosophila melanogaster GlcNAc-1-phosphotransferase
Map data
Sample
  • Complex: gnpt
    • Protein or peptide: FI02838p
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


N-glycan processing to lysosome / UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase activity / carbohydrate phosphorylation / membrane => GO:0016020 / Golgi apparatus
Similarity search - Function
Stealth protein CR2, conserved region 2 / Stealth protein CR4, conserved region 4 / Stealth protein CR3, conserved region 3 / Stealth protein CR1, conserved region 1 / Stealth protein CR2, conserved region 2 / Stealth protein CR1, conserved region 1 / Stealth protein CR3, conserved region 3 / Stealth protein CR4, conserved region 4 / Notch-like domain superfamily / LNR (Lin-12/Notch) repeat profile. ...Stealth protein CR2, conserved region 2 / Stealth protein CR4, conserved region 4 / Stealth protein CR3, conserved region 3 / Stealth protein CR1, conserved region 1 / Stealth protein CR2, conserved region 2 / Stealth protein CR1, conserved region 1 / Stealth protein CR3, conserved region 3 / Stealth protein CR4, conserved region 4 / Notch-like domain superfamily / LNR (Lin-12/Notch) repeat profile. / LNR domain / Notch domain / Domain found in Notch and Lin-12
Similarity search - Domain/homology
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsDu S / Xiao J / Guopeng W
CitationJournal: J Biol Chem / Year: 2022
Title: Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport.
Authors: Shuo Du / Guopeng Wang / Zhiying Zhang / Chengying Ma / Ning Gao / Junyu Xiao /
Abstract: GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are ...GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.
History
DepositionJan 19, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateAug 17, 2022-
Current statusAug 17, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.01
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  • Surface view with fitted model
  • Atomic models: PDB-7dxi
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30910.png

Downloads & links

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Map

FileDownload / File: emd_30910.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.6516 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.046524677 - 0.07315917
Average (Standard dev.)0.000116832496 (±0.0018452738)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 208.512 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.65160.65160.6516
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z208.512208.512208.512
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0470.0730.000

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Supplemental data

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Mask #1

Fileemd_30910_msk_1.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 : gnpt

EntireName: gnpt
Components
  • Complex: gnpt
    • Protein or peptide: FI02838p
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: gnpt

SupramoleculeName: gnpt / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #1: FI02838p

MacromoleculeName: FI02838p / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 67.9255 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: EEGQTGGFSS ACTAIDAVYT WVNGSDPNFI EDIRRFDDKY DPSRFDDKNE LRYSLRSLEK HAAWIRHVYI VTNGQIPSWL DLSYERVTV VPHEVLAPDP DQLPTFSSSA IETFLHRIPK LSKRFLYLND DIFLGAPLYP EDLYTEAEGV RVYQAWMVPD C ALDCPWTY ...String:
EEGQTGGFSS ACTAIDAVYT WVNGSDPNFI EDIRRFDDKY DPSRFDDKNE LRYSLRSLEK HAAWIRHVYI VTNGQIPSWL DLSYERVTV VPHEVLAPDP DQLPTFSSSA IETFLHRIPK LSKRFLYLND DIFLGAPLYP EDLYTEAEGV RVYQAWMVPD C ALDCPWTY IGDGACDRHC NIDACQFDGG DCSETGPASD AHVIPPSKEV LEVQPAAVPQ SRVHRFPQMG LQKLFRRSSA NF KDVMRHR NVSTLKELRR IVERFNKAKL MSLNPELETS SSEPQTTQRH GLRKEDFKSS TDIYSHSLIA TNMLLNRAYG FKA RHVLAH VGFLIDKDIV EAMQRRFHQQ ILDTAHQRFR APTDLQYAFA YYSFLMSETK VMSVEEIFDE FDTDGSATWS DREV RTFLT RIYQPPLDWS AMRYFEEVVQ NCTRNLGMHL KVDTVEHSTL VYERYEDSNL PTITRDLVVR CPLLAEALAA NFAVR PKYN FHVSPKRTSH SNFMMLTSNL TEVVESLDRL RRNPRKFNCI NDNLDANRGE DNEMVRHLLE DFYLSFFPRR SKFELP PQY RNRFESWRDF QRWKRRKR

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average exposure time: 3.2 sec. / Average electron dose: 60.29 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 131301

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