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- EMDB-53319: DNA polymerase with inhibitor #2 -

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Basic information

Entry
Database: EMDB / ID: EMD-53319
TitleDNA polymerase with inhibitor #2
Map dataexperimental map
Sample
  • Complex: DNA polymerase with DNA and inhibitor
    • Protein or peptide: DNA polymerase III PolC-type
    • DNA: DNA (5'-D(P*TP*AP*A)-3')
    • DNA: DNA (5'-D(P*GP*GP*TP*GP*CP*GP*CP*AP*T)-3')
    • DNA: DNA (5'-D(P*CP*CP*AP*TP*GP*CP*GP*CP*AP*CP*C)-3')
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: Ibezapolstat
  • Ligand: water
KeywordsDNA polymerase / inhibitor complex / DNA BINDING PROTEIN
Function / homology
Function and homology information


3'-5' exonuclease activity / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding / cytoplasm
Similarity search - Function
DNA polymerase III PolC-like, N-terminal domain II / DNA polymerase III PolC-type, N-terminal domain I / DNA polymerase III polC-type N-terminus II / DNA polymerase III polC-type N-terminus I / DNA polymerase III, alpha subunit, Gram-positive type / PolC, middle finger subdomain superfamily / DNA polymerase III epsilon subunit, exonuclease domain / DNA polymerase III, alpha subunit / Bacterial DNA polymerase III, alpha subunit, NTPase domain / DNA polymerase, helix-hairpin-helix motif ...DNA polymerase III PolC-like, N-terminal domain II / DNA polymerase III PolC-type, N-terminal domain I / DNA polymerase III polC-type N-terminus II / DNA polymerase III polC-type N-terminus I / DNA polymerase III, alpha subunit, Gram-positive type / PolC, middle finger subdomain superfamily / DNA polymerase III epsilon subunit, exonuclease domain / DNA polymerase III, alpha subunit / Bacterial DNA polymerase III, alpha subunit, NTPase domain / DNA polymerase, helix-hairpin-helix motif / DNA polymerase III alpha subunit finger domain / Bacterial DNA polymerase III alpha NTPase domain / Helix-hairpin-helix motif / Bacterial DNA polymerase III alpha subunit finger domain / PHP domain / PHP domain / Polymerase/histidinol phosphatase, N-terminal / DNA polymerase alpha chain like domain / Polymerase/histidinol phosphatase-like / Exonuclease / Exonuclease, RNase T/DNA polymerase III / EXOIII / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA polymerase III PolC-type
Similarity search - Component
Biological speciesEnterococcus faecium (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLamers MH / Urem M / Smits WK
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Other governmentPOLSTOP2 Netherlands
CitationJournal: Nat Commun / Year: 2025
Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens.
Authors: Mia Urem / Annemieke H Friggen / Nina Musch / Michael H Silverman / Christopher J Swain / Michael R Barbachyn / Lawrence I Mortin / Xiang Yu / Robert J DeLuccia / Meindert H Lamers / Wiep Klaas Smits /
Abstract: Infections with antimicrobial resistant pathogens are a major threat to human health. Inhibitors of the replicative polymerase PolC are a promising novel class of antimicrobials against Gram-positive ...Infections with antimicrobial resistant pathogens are a major threat to human health. Inhibitors of the replicative polymerase PolC are a promising novel class of antimicrobials against Gram-positive pathogens, but the structural basis for their activity remains unknown. The first-in-class PolC-targeting antimicrobial, ibezapolstat, is a guanine analogue in late-stage clinical development for the treatment of Clostridioides difficile infections, and related inhibitors are being developed for systemic treatment of infections with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Here, we present the cryo-electron microscopy structures of Enterococcus faecium PolC bound to DNA and in complex with ibezapolstat or the previously-undescribed inhibitor ACX-801. Both inhibitors form base-pairing interactions with the DNA in the active site, thereby competing with incoming dGTP nucleotides. We identify a crucial susceptibility determinant in PolC that is conserved in other organisms, such as C. difficile. This is explained by an unusual non-planar conformation of the inhibitors that induce a binding pocket in PolC. By combining structural, biochemical, bioinformatic and genetic analyses, this work lays the foundation for the rational development of an innovative class of antimicrobials against Gram-positive priority pathogens.
History
DepositionApr 3, 2025-
Header (metadata) releaseSep 24, 2025-
Map releaseSep 24, 2025-
UpdateApr 8, 2026-
Current statusApr 8, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53319.png

Downloads & links

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Map

FileDownload / File: emd_53319.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationexperimental map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 256 pix.
= 214.016 Å		0.84 Å/pix.
x 256 pix.
= 214.016 Å		0.84 Å/pix.
x 256 pix.
= 214.016 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0116
Minimum - Maximum-0.04290566 - 0.06435433
Average (Standard dev.)0.000108199885 (±0.0016266262)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 214.016 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: halfmap 1

Fileemd_53319_half_map_1.map
Annotationhalfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap 2

Fileemd_53319_half_map_2.map
Annotationhalfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DNA polymerase with DNA and inhibitor

EntireName: DNA polymerase with DNA and inhibitor
Components
  • Complex: DNA polymerase with DNA and inhibitor
    • Protein or peptide: DNA polymerase III PolC-type
    • DNA: DNA (5'-D(P*TP*AP*A)-3')
    • DNA: DNA (5'-D(P*GP*GP*TP*GP*CP*GP*CP*AP*T)-3')
    • DNA: DNA (5'-D(P*CP*CP*AP*TP*GP*CP*GP*CP*AP*CP*C)-3')
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: Ibezapolstat
  • Ligand: water

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Supramolecule #1: DNA polymerase with DNA and inhibitor

SupramoleculeName: DNA polymerase with DNA and inhibitor / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Enterococcus faecium (bacteria)
Molecular weightTheoretical: 166 KDa

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Macromolecule #1: DNA polymerase III PolC-type

MacromoleculeName: DNA polymerase III PolC-type / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Enterococcus faecium (bacteria)
Molecular weightTheoretical: 166.595766 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MHMSEKRDLF EKLLEQIQLE EAEKNHPLLT AGEMDSVVVH RKSRLWEFTL HFSKILPIML YRSLTQHLT IAFKDIAQVK LNILADDQTF DEQLLQDYWA QALENQQCDT PLAQQVLKTQ VPVIKDRKVI LPIDSTGAIS Y LKQQYLPL ...String:
MGSSHHHHHH SSGLVPRGSH MHMSEKRDLF EKLLEQIQLE EAEKNHPLLT AGEMDSVVVH RKSRLWEFTL HFSKILPIML YRSLTQHLT IAFKDIAQVK LNILADDQTF DEQLLQDYWA QALENQQCDT PLAQQVLKTQ VPVIKDRKVI LPIDSTGAIS Y LKQQYLPL VEELFVSYGF PKFRIEPEVD EQQAERVLKL FEERKQEQAE AFMKQAAESL VVHEQKKKER KEQSPALEGP IH IQLGRNI PADEPTTPMI SIVEEERRVT LEGYVFDKEV RELRSKRKIL TLKITDYTSS FIVKKFSNGE KDEQVFDAIS VGS WLKVRG SIQEDTFVRD LVMNAQDIIE VKHTPRKDYA PEGEKRVELH VHSNMSTMDA TNSISDLVAQ AGKWGHRAIA ITDH GGAQA FPEAHSAGKK AGVKILYGVE ANVVDDGVPI AYNDAHEALS EATYVVFAVA TTGLSAVYDT IIELAAVKMY KGNVI ESFD EFIDPGHPLS RTTVDLTGIT DGMVRGSKSE EEVLRMFLEF SKDTILVAHN AAFDMGFLNT SYARYGIPEA ANPVID TLE LARYLYPQFK RFGLGVLSKK FGVSLEQHHR AIYDAEATGH LAWIFVKEAM DNHNMLYHDQ LNEHIGEGDS YKRARPF HV TILAKNQAGL KDLFKLISMS NVEYFERVPR IPRSQLKKMR ENLLIGSACD KGEIFEAMMQ KGVEEARNRA KFYDYIEV M PKAVYAPLIE QELVKNEHDL EEIIQNLVEI GKSLDKIVVA TGNVHYLNEE DAIYRKILIN SMGGANPLNR HSLPDVHFR TTDEMLTAFH FLGEETAKEI VVENTNKIAD ICEEVIPVKD ELYTPKIPGS EDEISELSYT KAKQMYGDPL PEIIQKRLKK ELNSINGNG FSVIYLIAQK LVHKSNEDGY LVGSRGSVGS SFVATMTGIT EVNPLAPHYY CPECQYSEFF EDGTYGSGFD M PEKQCPKC GARLNKDGHD IPFETFLGFH GDKVPDIDLN FSGDYQAEAH NYTKVLFGED YVYRAGTIGT VADKTAYGYV KG YERDNNL QFRSAEVDRL AKGATGVKRT TGQHPGGIIV IPDYMDVYDF TPIQYPADDQ NSEWKTTHFD FHSIHDNVLK LDI LGHDDP TVIRMLQDLS GIDPQTIPTD DPEVMRIFAG PEVLGVSQEQ IYSKTGTLGI PEFGTRFVRG MLEETHPTTF AELL QISGL SHGTDVWLGN AEELIRRGDA TLAEVIGCRD DIMVYLIHAG LDSGMAFKIM ETVRKGQWNK IPDELRETYL SAMKE NNVP DWYIDSCSKI KYMFPKAHAA AYVLMALRVA YFKVYFPILY YCAYFSVRAD DFDLVSMCKG KDAVKQAMKE ITDKGL DAS VKEKNQLTVL ELANEMLERG FKFGMIDLYK SDAVNFVIEG DTLIAPFRAV PSLGTNVAKQ IVEARKDGPF LSKEDLA TR GKVSKTLIEY MNDNGVLKDL PDENQLSLFD ML

UniProtKB: DNA polymerase III PolC-type

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Macromolecule #2: DNA (5'-D(P*TP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*TP*AP*A)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 885.649 Da
SequenceString:
(DT)(DA)(DA)

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Macromolecule #3: DNA (5'-D(P*GP*GP*TP*GP*CP*GP*CP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*GP*GP*TP*GP*CP*GP*CP*AP*T)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10.179559 KDa
SequenceString:
(DT)(DA)(DG)(DG)(DA)(DC)(DG)(DA)(DA)(DG) (DG)(DA)(DC)(DT)(DC)(DC)(DC)(DA)(DA)(DC) (DT)(DT)(DT)(DA)(DG)(DG)(DT)(DG)(DC) (DG)(DC)(DA)(DT)

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Macromolecule #4: DNA (5'-D(P*CP*CP*AP*TP*GP*CP*GP*CP*AP*CP*C)-3')

MacromoleculeName: DNA (5'-D(P*CP*CP*AP*TP*GP*CP*GP*CP*AP*CP*C)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 12.724144 KDa
SequenceString:
(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DA) (DT)(DG)(DC)(DG)(DC)(DA)(DC)(DC)(DT)(DA) (DA)(DA)(DG)(DT)(DT)(DG)(DG)(DG)(DA) (DG)(DT)(DC)(DC)(DT)(DT)(DC)(DG)(DT)(DC) (DC) (DT)(DA)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: Ibezapolstat

MacromoleculeName: Ibezapolstat / type: ligand / ID: 7 / Number of copies: 1 / Formula: A1I9T
Molecular weightTheoretical: 423.296 Da

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 1 / 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

Concentration0.3 mg/mL
BufferpH: 8
Details: 50 mM HEPES pH 8.0 50 mM NaCl 7.5 mM MgCl2 2 mM DTT 0.05% Tween 20.
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 193 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 330001
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING

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Atomic model buiding 1

Initial modelPDB ID:

cxw7


Chain - Chain ID: A / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-9qrl:
DNA polymerase with inhibitor #2

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