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- EMDB-44511: Structure of human GlcNAc-1-phosphotransferase complexed with the... -

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Basic information

Entry
Database: EMDB / ID: EMD-44511
TitleStructure of human GlcNAc-1-phosphotransferase complexed with the donor substrate UDP-GlcNAc
Map data
Sample
  • Complex: GlcNAc-1-phosphotransferase
    • Protein or peptide: N-acetylglucosamine-1-phosphotransferase subunits alpha/beta
  • Ligand: CALCIUM ION
  • Ligand: MAGNESIUM ION
  • Ligand: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsGlcNAc-1-phosphotransferase / lysosomal hydrolases / mannose 6-phosphate trafficking pathway / TRANSFERASE
Function / homology
Function and homology information


UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase / N-glycan processing to lysosome / UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase activity / secretion of lysosomal enzymes / carbohydrate phosphorylation / lysosome organization / Golgi membrane / calcium ion binding / Golgi apparatus
Similarity search - Function
N-acetylglucosamine-1-phosphotransferase subunit alpha/beta, regulatory domain / Putative GlcNAc-1 phosphotransferase regulatory domain / 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 ...N-acetylglucosamine-1-phosphotransferase subunit alpha/beta, regulatory domain / Putative GlcNAc-1 phosphotransferase regulatory domain / 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 / DMAP1-binding Domain / DMAP1-binding Domain / DMAP1-binding domain / DMAP1-binding domain profile. / Notch-like domain superfamily / LNR domain / LNR (Lin-12/Notch) repeat profile. / Notch domain / Domain found in Notch and Lin-12 / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain
Similarity search - Domain/homology
N-acetylglucosamine-1-phosphotransferase subunits alpha/beta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLi H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS127292 United States
CitationJournal: J Biol Chem / Year: 2024
Title: Structure of a truncated human GlcNAc-1-phosphotransferase variant reveals the basis for its hyperactivity.
Authors: Hua Li / Balraj Doray / Benjamin C Jennings / Wang-Sik Lee / Lin Liu / Stuart Kornfeld / Huilin Li /
Abstract: Mutations that cause loss of function of GlcNAc-1-phosphotransferase (PTase) lead to the lysosomal storage disorder mucolipidosis II. PTase is the key enzyme of the mannose 6-phosphate (M6P) ...Mutations that cause loss of function of GlcNAc-1-phosphotransferase (PTase) lead to the lysosomal storage disorder mucolipidosis II. PTase is the key enzyme of the mannose 6-phosphate (M6P) targeting system that is responsible for tagging lysosomal hydrolases with the M6P moiety for their delivery to the lysosome. We had previously generated a truncated hyperactive form of PTase termed S1S3 which was shown to notably increase the phosphorylation level of secreted lysosomal enzymes and enhance their uptake by cells. Here, we report the 3.4 Å cryo-EM structure of soluble S1S3 lacking both transmembrane domains and cytosolic tails. The structure reveals a high degree of conservation of the catalytic core to full-length PTase. In this dimeric structure, the EF-hand of one protomer is observed interacting with the conserved region four of the other. In addition, we present a high-quality EM 3D map of the UDP-GlcNAc bound form of the full-length soluble protein showing the key molecular interactions between the nucleotide sugar donor and side chain amino acids of the protein. Finally, although the domain organization of S1S3 is very similar to that of the Drosophila melanogaster (fruit fly) PTase homolog, we establish that the latter does not act on lysosomal hydrolases.
History
DepositionApr 18, 2024-
Header (metadata) releaseFeb 26, 2025-
Map releaseFeb 26, 2025-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44511.png

Downloads & links

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Map

FileDownload / File: emd_44511.map.gz / Format: CCP4 / Size: 125 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 320 pix.
= 264.96 Å		0.83 Å/pix.
x 320 pix.
= 264.96 Å		0.83 Å/pix.
x 320 pix.
= 264.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.065193824 - 0.09365847
Average (Standard dev.)0.000060299302 (±0.0014411783)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44511_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44511_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44511_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : GlcNAc-1-phosphotransferase

EntireName: GlcNAc-1-phosphotransferase
Components
  • Complex: GlcNAc-1-phosphotransferase
    • Protein or peptide: N-acetylglucosamine-1-phosphotransferase subunits alpha/beta
  • Ligand: CALCIUM ION
  • Ligand: MAGNESIUM ION
  • Ligand: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: GlcNAc-1-phosphotransferase

SupramoleculeName: GlcNAc-1-phosphotransferase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 268 KDa

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Macromolecule #1: N-acetylglucosamine-1-phosphotransferase subunits alpha/beta

MacromoleculeName: N-acetylglucosamine-1-phosphotransferase subunits alpha/beta
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 134.7875 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DEDQVDPRLI DGKWSRDQYH VLFDSYRDNI AGKSFQNRLC LPMPIDVVYT WVNGTDLELL KELQQVREQM EEEQKAMREI LGKNTTEPT KKSEKQLECL LTHCIKVPML VLDPALPANI TLKDLPSLYP SFHSASDIFN VAKPKNPSTN VSVVVFDSTK D VEDAHSGL ...String:
DEDQVDPRLI DGKWSRDQYH VLFDSYRDNI AGKSFQNRLC LPMPIDVVYT WVNGTDLELL KELQQVREQM EEEQKAMREI LGKNTTEPT KKSEKQLECL LTHCIKVPML VLDPALPANI TLKDLPSLYP SFHSASDIFN VAKPKNPSTN VSVVVFDSTK D VEDAHSGL LKGNSRQTVW RGYLTTDKEV PGLVLMQDLA FLSGFPPTFK ETNQLKTKLP ENLSSKVKLL QLYSEASVAL LK LNNPKDF QELNKQTKKN MTIDGKELTI SPAYLLWDLS AISQSKQDED ISASRFEDNE ELRYSLRSIE RHAPWVRNIF IVT NGQIPS WLNLDNPRVT IVTHQDVFRN LSHLPTFSSP AIESHIHRIE GLSQKFIYLN DDVMFGKDVW PDDFYSHSKG QKVY LTWPV PNCAEGCPGS WIKDGYCDKA CNNSACDWDG GDCSGNSGGS RYIAGGGGTG SIGVGQPWQF GGGINSVSYC NQGCA NSWL ADKFCDQACN VLSCGFDAGD CGQDHFHELY KVILLPNQTH YIIPKGECLP YFSFAEVAKR GVEGAYSDNP IIRHAS IAN KWKTIHLIMH SGMNATTIHF NLTFQNTNDE EFKMQITVEV DTREGPKLNS TAQKGYENLV SPITLLPEAE ILFEDIP KE KRFPKFKRHD VNSTRRAQEE VKIPLVNISL LPKDAQLSLN TLDLQLEHGD ITLKGYNLSK SALLRSFLMN SQHAKIKN Q AIITDETNDS LVAPQEKQVH KSILPNSLGV SERLQRLTFP AVSVKVNGHD QGQNPPLDLE TTARFRVETH TQKTIGGNV TKEKPPSLIV PLESQMTKEK KITGKEKENS RMEENAENHI GVTEVLLGRK LQHYTDSYLG FLPWEKKKYF QDLLDEEESL KTQLAYFTD SKNRARYKRD TFADSLRYVN KILNSKFGFT SRKVPAHMPH MIDRIVMQEL QDMFPEEFDK TSFHKVRHSE D MQFAFSYF YYLMSAVQPL NISQVFDEVD TDQSGVLSDR EIRTLATRIH ELPLSLQDLT GLEHMLINCS KMLPADITQL NN IPPTQES YYDPNLPPVT KSLVTNCKPV TDKIHKAYKD KNKYRFEIMG EEEIAFKMIR TNVSHVVGQL DDIRKNPRKF VCL NDNIDH NHKDAQTVKA VLRDFYESMF PIPSQFELPR EYRNRFLHMH ELQEWRAYRD KLK

UniProtKB: N-acetylglucosamine-1-phosphotransferase subunits alpha/beta

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE

MacromoleculeName: URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE / type: ligand / ID: 5 / Number of copies: 2 / Formula: UD1
Molecular weightTheoretical: 607.354 Da
Chemical component information

ChemComp-UD1:
URIDINE-DIPHOSPHATE-N-ACETYLGLUCOSAMINE

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 6 / 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

Concentration0.15 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
75.0 mMKClpotassium chloride
20.0 mMHEPES
10.0 mM2-mercaptoethanol
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 299 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 193.0 K / Max: 193.0 K
Alignment procedureComa free - Residual tilt: 0.05 mrad
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 24130 / Average exposure time: 1.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.0 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: 2859876
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 876204
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7s05


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-9bgf:
Structure of human GlcNAc-1-phosphotransferase complexed with the donor substrate UDP-GlcNAc

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