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- EMDB-74451: Cryo-EM structure of human UDP-N-acetylglucosamine-dolichyl-phosp... -

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Basic information

Entry
Database: EMDB / ID: EMD-74451
TitleCryo-EM structure of human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB, composite map
Map dataComposite Map from Local Refinements of Chains A and B
Sample
  • Complex: Human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB
    • Protein or peptide: UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase
  • Ligand: 4-({(2S,3S)-1-amino-3-{[(2S,3R,4S,5R)-5-(aminomethyl)-3,4-dihydroxyoxolan-2-yl]oxy}-3-[(2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxyoxolan-2-yl]-1-oxopropan-2-yl}amino)-N-[(4-{4-[4-(trifluoromethoxy)phenoxy]piperidin-1-yl}phenyl)methyl]butanamide (non-preferred name)
  • Ligand: (2R)-3-{[(S)-[(2R)-2,3-dihydroxypropoxy](hydroxy)phosphoryl]oxy}-2-{[(9Z)-octadec-9-enoyl]oxy}propyl (9Z)-octadec-9-enoate
  • Ligand: water
Keywordspolyprenyl-phosphate N-acetylhexosamine phosphate transferase / phosphoglycosyltransferase / membrane protein / TRANSFERASE
Function / homology
Function and homology information


Biosynthesis of the N-glycan precursor (dolichol lipid-linked oligosaccharide, LLO) and transfer to a nascent protein / Defective DPAGT1 causes CDG-1j, CMSTA2 / UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase / UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase activity / UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase activity / dolichol-linked oligosaccharide biosynthetic process / protein N-linked glycosylation / glycosyltransferase activity / intracellular membrane-bounded organelle / endoplasmic reticulum membrane ...Biosynthesis of the N-glycan precursor (dolichol lipid-linked oligosaccharide, LLO) and transfer to a nascent protein / Defective DPAGT1 causes CDG-1j, CMSTA2 / UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase / UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase activity / UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase activity / dolichol-linked oligosaccharide biosynthetic process / protein N-linked glycosylation / glycosyltransferase activity / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / metal ion binding / identical protein binding / membrane
Similarity search - Function
UDP-GlcNAc-dolichyl-phosphate GlcNAc phosphotransferase / : / DPAGT1 insertion domain / Glycosyl transferase, family 4 / Glycosyl transferase family 4
Similarity search - Domain/homology
UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKirsh JM / Ochoa JM / Soroush-Pejrimovsky MT / Kaudeer BY / Clemons WM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM114611 United States
The G. Harold and Leila Y. Mathers Foundation United States
Chan Zuckerberg Initiative United States
CitationJournal: bioRxiv / Year: 2025
Title: Structures of bacterial and human phosphoglycosyltransferases bound to a common inhibitor inform selective therapeutics.
Authors: Beebee Yusrah Kaudeer / Jacob M Kirsh / Katsuhiko Mitachi / Jessica M Ochoa / Marie-Therese Soroush-Pejrimovsky / Yancheng E Li / Vy N Nguyen / Michio Kurosu / William M Clemons /
Abstract: Glycoconjugates facilitate myriad biological processes, including cell-cell recognition and immune response, and they are generated by enzymes that transfer glycans. The orthologs MraY and DPAGT1 are ...Glycoconjugates facilitate myriad biological processes, including cell-cell recognition and immune response, and they are generated by enzymes that transfer glycans. The orthologs MraY and DPAGT1 are dimeric phosphoglycosyltransferases involved in oligosaccharide biosynthesis for either bacterial peptidoglycan or eukaryotic -linked glycans, respectively. Both enzymes play central regulatory roles, making them attractive targets for antibacterial and anticancer therapies. In our prior studies, a muraymycin A1-derived inhibitor termed APPB (aminouridyl phenoxypiperidinbenzyl butanamide) was developed. It exhibits sub-100 nM IC50 values against both MraY and DPAGT1 and has demonstrated efficacy against DPAGT1-dependent cancers, making it an excellent starting point for next-generation small molecules. To guide inhibitor development, we determined cryo-EM structures of APPB bound to MraY or DPAGT1 at 2.9 Å resolution using single-particle analysis. The structures reveal that APPB, composed of a nucleoside, a central amide, and a lipid-mimetic, adopts two conformations in each protein, which correlate with local hydrogen-bonding contacts of the central amide carbonyl. Examination of the amide carbonyl environments guides conformer selection for future DPAGT1-targeting anticancer agents. Further, comparisons of APPB-bound geometries and nucleoside interactions inform opportunities for antibacterial agents targeting MraY. Overall, our study provides design principles for MraY- or DPAGT1-specific drugs and motivates the utility of simultaneously characterizing inhibitor-bound orthologs for selective therapeutics.
History
DepositionDec 14, 2025-
Header (metadata) releaseFeb 4, 2026-
Map releaseFeb 4, 2026-
UpdateFeb 4, 2026-
Current statusFeb 4, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_74451.png

Downloads & links

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Map

FileDownload / File: emd_74451.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite Map from Local Refinements of Chains A and B
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0000261
Minimum - Maximum-0.3672262 - 0.63463753
Average (Standard dev.)0.00021799901 (±0.0077202264)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosam...

EntireName: Human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB
Components
  • Complex: Human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB
    • Protein or peptide: UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase
  • Ligand: 4-({(2S,3S)-1-amino-3-{[(2S,3R,4S,5R)-5-(aminomethyl)-3,4-dihydroxyoxolan-2-yl]oxy}-3-[(2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxyoxolan-2-yl]-1-oxopropan-2-yl}amino)-N-[(4-{4-[4-(trifluoromethoxy)phenoxy]piperidin-1-yl}phenyl)methyl]butanamide (non-preferred name)
  • Ligand: (2R)-3-{[(S)-[(2R)-2,3-dihydroxypropoxy](hydroxy)phosphoryl]oxy}-2-{[(9Z)-octadec-9-enoyl]oxy}propyl (9Z)-octadec-9-enoate
  • Ligand: water

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Supramolecule #1: Human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosam...

SupramoleculeName: Human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: DPAGT1 incubated with small molecule APPB for one hour on ice
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100.312 KDa

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Macromolecule #1: UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosamineph...

MacromoleculeName: UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase
type: protein_or_peptide / ID: 1
Details: Expressed as fusion protein with C-terminal TEV cleavage site followed by GFP; GFP was cleaved using TEV protease
Number of copies: 2 / Enantiomer: LEVO
EC number: UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.312938 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MWAFSELPMP LLINLIVSLL GFVATVTLIP AFRGHFIAAR LCGQDLNKTS RQQIPESQGV ISGAVFLIIL FCFIPFPFLN CFVKEQCKA FPHHEFVALI GALLAICCMI FLGFADDVLN LRWRHKLLLP TAASLPLLMV YFTNFGNTTI VVPKPFRPIL G LHLDLGIL ...String:
MWAFSELPMP LLINLIVSLL GFVATVTLIP AFRGHFIAAR LCGQDLNKTS RQQIPESQGV ISGAVFLIIL FCFIPFPFLN CFVKEQCKA FPHHEFVALI GALLAICCMI FLGFADDVLN LRWRHKLLLP TAASLPLLMV YFTNFGNTTI VVPKPFRPIL G LHLDLGIL YYVYMGLLAV FCTNAINILA GINGLEAGQS LVISASIIVF NLVELEGDCR DDHVFSLYFM IPFFFTTLGL LY HNWYPSR VFVGDTFCYF AGMTFAVVGI LGHFSKTMLL FFMPQVFNFL YSLPQLLHII PCPRHRIPRL NIKTGKLEMS YSK FKTKSL SFLGTFILKV AESLQLVTVH QSETEDGEFT ECNNMTLINL LLKVLGPIHE RNLTLLLLLL QILGSAITFS IRYQ LVRLF YDVGSSASEN LYFQ

UniProtKB: UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase

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Macromolecule #2: 4-({(2S,3S)-1-amino-3-{[(2S,3R,4S,5R)-5-(aminomethyl)-3,4-dihydro...

MacromoleculeName: 4-({(2S,3S)-1-amino-3-{[(2S,3R,4S,5R)-5-(aminomethyl)-3,4-dihydroxyoxolan-2-yl]oxy}-3-[(2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxyoxolan-2-yl]-1-oxopropan-2-yl}amino) ...Name: 4-({(2S,3S)-1-amino-3-{[(2S,3R,4S,5R)-5-(aminomethyl)-3,4-dihydroxyoxolan-2-yl]oxy}-3-[(2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxyoxolan-2-yl]-1-oxopropan-2-yl}amino)-N-[(4-{4-[4-(trifluoromethoxy)phenoxy]piperidin-1-yl}phenyl)methyl]butanamide (non-preferred name)
type: ligand / ID: 2 / Number of copies: 2 / Formula: A1C3G
Molecular weightTheoretical: 881.849 Da

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Macromolecule #3: (2R)-3-{[(S)-[(2R)-2,3-dihydroxypropoxy](hydroxy)phosphoryl]oxy}-...

MacromoleculeName: (2R)-3-{[(S)-[(2R)-2,3-dihydroxypropoxy](hydroxy)phosphoryl]oxy}-2-{[(9Z)-octadec-9-enoyl]oxy}propyl (9Z)-octadec-9-enoate
type: ligand / ID: 3 / Number of copies: 2 / Formula: A1C3H
Molecular weightTheoretical: 775.044 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 80 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration4.0 mg/mL
BufferpH: 7.5
Details: 50 mM HEPES pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.022% (w/v) DDM, and 2 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 75 sec. / Pretreatment - Pressure: 30.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 2-3 uL of sample distributed on grid with blot force +8 and blot time 3 seconds.
DetailsDPAGT1 incubated with 0.4 mM APPB on ice for 1 hour

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: AlphaFold2 prediction of dimeric apo DPAGT1
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 82209
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 79.7
Output model

PDB-9znn:
Cryo-EM structure of human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB

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