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- PDB-9zno: Cryo-EM structure of Hydrogenivirga sp. MraY in complex with APPB -

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Entry
Database: PDB / ID: 9zno
TitleCryo-EM structure of Hydrogenivirga sp. MraY in complex with APPB
ComponentsPhospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)
KeywordsTRANSFERASE / polyprenyl-phosphate N-acetylhexosamine phosphate transferase / phosphoglycosyltransferases / membrane protein
Function / homology:
Function and homology information
Biological speciesHydrogenivirga sp. 128-5-R1-1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsKaudeer, 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: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / 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

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Assembly

Deposited unit
A: Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)
B: Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)83,6894
Polymers81,9252
Non-polymers1,7642
Water00
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 Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)


Mass: 40962.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Expressed this fusion protein with N-terminal 6xHis tag.
Source: (gene. exp.) Hydrogenivirga sp. 128-5-R1-1 (bacteria)
Gene: mraY / Plasmid: pET22b / Production host: Escherichia coli K-12 (bacteria)
Strain (production host): NiCo21 (DE3) pLEMO (DE3) NiCo21 (DE3) pLEMO (DE3)
#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 / Feature type: SUBJECT OF INVESTIGATION
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: Complex of HyMraY with APPB / Type: COMPLEX
Details: MraY incubated with small molecule APPB for 1 hour on ice
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.083631 MDa / Experimental value: NO
Source (natural)Organism: Hydrogenivirga sp. 128-5-R1-1 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria) / Cell: NiCo21 (DE3) pLEMO (DE3) / Plasmid: pET22b
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 75 mM NaCl, 5% glycerol, 0.15% DM and 10 mM MgCl2
SpecimenConc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: MraY 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: 3 uL sample 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 magnification: 130000 X / 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
2PHENIX1.21.1_5286model refinement
13cryoSPARC4.7.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107945 / Symmetry type: POINT
Atomic model buildingB value: 109.8 / Protocol: RIGID BODY FIT / Space: REAL
RefinementHighest resolution: 2.91 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035590
ELECTRON MICROSCOPYf_angle_d0.587608
ELECTRON MICROSCOPYf_dihedral_angle_d11.048912
ELECTRON MICROSCOPYf_chiral_restr0.037908
ELECTRON MICROSCOPYf_plane_restr0.008914

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