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- EMDB-74452: Cryo-EM structure of Hydrogenivirga sp. MraY in complex with APPB -

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Basic information

Entry
Database: EMDB / ID: EMD-74452
TitleCryo-EM structure of Hydrogenivirga sp. MraY in complex with APPB
Map dataSharpened map
Sample
  • Complex: Complex of HyMraY with APPB
    • Protein or peptide: Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)
  • 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)
Keywordspolyprenyl-phosphate N-acetylhexosamine phosphate transferase / phosphoglycosyltransferases / membrane protein / TRANSFERASE
Biological speciesHydrogenivirga sp. 128-5-R1-1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsKaudeer 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_74452.png

Downloads & links

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Map

FileDownload / File: emd_74452.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0177
Minimum - Maximum-0.3801464 - 0.5663178
Average (Standard dev.)0.00025342937 (±0.008595401)
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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Additional map: Unsharpened map

Fileemd_74452_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_74452_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_74452_half_map_2.map
Projections & Slices
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Sample components

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Entire : Complex of HyMraY with APPB

EntireName: Complex of HyMraY with APPB
Components
  • Complex: Complex of HyMraY with APPB
    • Protein or peptide: Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)
  • 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)

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Supramolecule #1: Complex of HyMraY with APPB

SupramoleculeName: Complex of HyMraY with APPB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: MraY incubated with small molecule APPB for 1 hour on ice
Source (natural)Organism: Hydrogenivirga sp. 128-5-R1-1 (bacteria)
Molecular weightTheoretical: 83.631 KDa

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Macromolecule #1: Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)

MacromoleculeName: Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY)
type: protein_or_peptide / ID: 1
Details: Expressed this fusion protein with N-terminal 6xHis tag.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Hydrogenivirga sp. 128-5-R1-1 (bacteria)
Molecular weightTheoretical: 40.962703 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: HHHHHHMIYH LAILLREHFF AFNVLKYITF RSFTAILLAF FITLILSPTF MKKFAKIQRL FGGYVREYTP EHHESKKYTP TMGGVVIVT VILITSVLLM RLDIRYTWVL VFSTLSFALI GFVDDWIKLK NKKGLSIKAK LAFQMSFALA VSLLIFYWVG L ETKLYFPF ...String:
HHHHHHMIYH LAILLREHFF AFNVLKYITF RSFTAILLAF FITLILSPTF MKKFAKIQRL FGGYVREYTP EHHESKKYTP TMGGVVIVT VILITSVLLM RLDIRYTWVL VFSTLSFALI GFVDDWIKLK NKKGLSIKAK LAFQMSFALA VSLLIFYWVG L ETKLYFPF FKELTVDLGW LYIPFSMFII VGTANAVNLT DGLDGLAIGP SMTTATAFGV IAYVVGHSKI AQYLGVPHVP YA GEITVFC FAIIGAGLGF LWFNTYPAQV FMGDVGALGL GAALATVSIM TKSEFLLAVA GGVFVFETVT VILQIIYFRA TGG KRLFRK APFHHHLEEK GLDEPKIVVR MWIVSALLAI VSVAMLKLR

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration12 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES pH 7.5, 75 mM NaCl, 5% glycerol, 0.15% DM and 10 mM MgCl2
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: 3 uL sample with blot force +8 and blot time 3 seconds.
DetailsMraY 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 / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: AlphaFold2 prediction of dimeric apo HyMraY
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 107945
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 109.8
Output model

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

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