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- PDB-9znn: Cryo-EM structure of human UDP-N-acetylglucosamine-dolichyl-phosp... -

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Basic information

Entry
Database: PDB / ID: 9znn
TitleCryo-EM structure of human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB
ComponentsUDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase
KeywordsTRANSFERASE / polyprenyl-phosphate N-acetylhexosamine phosphate transferase / phosphoglycosyltransferase / membrane protein
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKirsh, J.M. / Ochoa, J.M. / Soroush-Pejrimovsky, M.T. / Kaudeer, B.Y. / Clemons, W.M.
Funding support United States, 3items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase
B: UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,9406
Polymers94,6262
Non-polymers3,3144
Water1,44180
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein UDP-N-acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase / GlcNAc-1-P transferase / G1PT / GPT / N-acetylglucosamine-1-phosphate transferase


Mass: 47312.938 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Expressed as fusion protein with C-terminal TEV cleavage site followed by GFP; GFP was cleaved using TEV protease
Source: (gene. exp.) Homo sapiens (human) / Gene: DPAGT1, DPAGT2 / Plasmid: pHAGE2 / Cell line (production host): Expi293F / Organ (production host): Kidney (Embryonic) / Production host: Homo sapiens (human)
References: UniProt: Q9H3H5, UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase
#2: Chemical ChemComp-A1C3G / 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)


Mass: 881.849 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C39H50F3N7O13
#3: Chemical ChemComp-A1C3H / (2R)-3-{[(S)-[(2R)-2,3-dihydroxypropoxy](hydroxy)phosphoryl]oxy}-2-{[(9Z)-octadec-9-enoyl]oxy}propyl (9Z)-octadec-9-enoate


Mass: 775.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H79O10P
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 80 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) in complex with APPB
Type: COMPLEX
Details: DPAGT1 incubated with small molecule APPB for one hour on ice
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.100312 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F / Plasmid: pHAGE2
Buffer solutionpH: 7.5
Details: 50 mM HEPES pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.022% (w/v) DDM, and 2 mM TCEP
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: DPAGT1 incubated with 0.4 mM APPB on ice for 1 hour
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 2-3 uL of sample distributed on grid with blot force +8 and blot time 3 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.1particle selection
4cryoSPARC4.7.1CTF correction
9cryoSPARC4.7.1initial Euler assignment
10cryoSPARC4.7.1final Euler assignment
12cryoSPARC4.7.13D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82209 / Symmetry type: POINT
Atomic model buildingB value: 79.7 / Protocol: RIGID BODY FIT / Space: REAL
RefinementHighest resolution: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036556
ELECTRON MICROSCOPYf_angle_d0.5918901
ELECTRON MICROSCOPYf_dihedral_angle_d12.9741019
ELECTRON MICROSCOPYf_chiral_restr0.041042
ELECTRON MICROSCOPYf_plane_restr0.0071103

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