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- EMDB-31339: Cryo-EM structure of the Gp168-beta-clamp complex -

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Basic information

Entry
Database: EMDB / ID: EMD-31339
TitleCryo-EM structure of the Gp168-beta-clamp complex
Map dataCryo-EM structure of the Gp168-beta-clamp complex
Sample
  • Complex: Gp168-beta-clamp complex
    • Complex: Beta sliding clamp 2
      • Protein or peptide: Beta sliding clamp
    • Complex: Gp168
      • Protein or peptide: Sliding clamp inhibitor
Function / homology
Function and homology information


DNA polymerase III complex / 3'-5' exonuclease activity / DNA replication / DNA-directed DNA polymerase activity / DNA binding / cytoplasm
Similarity search - Function
DNA polymerase III, beta sliding clamp / DNA polymerase III, beta sliding clamp, N-terminal / DNA polymerase III, beta sliding clamp, C-terminal / DNA polymerase III, beta sliding clamp, central / DNA polymerase III beta subunit, N-terminal domain / DNA polymerase III beta subunit, central domain / DNA polymerase III beta subunit, C-terminal domain / DNA polymerase III beta subunit
Similarity search - Domain/homology
Sliding clamp inhibitor / Beta sliding clamp
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria) / Staphylococcus virus Twort
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiu B / Li S / Liu Y / Chen H / Hu Z / Wang Z / Gou L / Zhang L / Ma B / Wang H ...Liu B / Li S / Liu Y / Chen H / Hu Z / Wang Z / Gou L / Zhang L / Ma B / Wang H / Matthews S / Wang Y / Zhang K
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81871662 China
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Bacteriophage Twort protein Gp168 is a β-clamp inhibitor by occupying the DNA sliding channel.
Authors: Bing Liu / Shanshan Li / Yang Liu / Huan Chen / Zhenyue Hu / Zhihao Wang / Yimin Zhao / Lei Zhang / Biyun Ma / Hongliang Wang / Steve Matthews / Yawen Wang / Kaiming Zhang /
Abstract: Bacterial chromosome replication is mainly catalyzed by DNA polymerase III, whose beta subunits enable rapid processive DNA replication. Enabled by the clamp-loading complex, the two beta subunits ...Bacterial chromosome replication is mainly catalyzed by DNA polymerase III, whose beta subunits enable rapid processive DNA replication. Enabled by the clamp-loading complex, the two beta subunits form a ring-like clamp around DNA and keep the polymerase sliding along. Given the essential role of β-clamp, its inhibitors have been explored for antibacterial purposes. Similarly, β-clamp is an ideal target for bacteriophages to shut off host DNA synthesis during host takeover. The Gp168 protein of phage Twort is such an example, which binds to the β-clamp of Staphylococcus aureus and prevents it from loading onto DNA causing replication arrest. Here, we report a cryo-EM structure of the clamp-Gp168 complex at 3.2-Å resolution. In the structure of the complex, the Gp168 dimer occupies the DNA sliding channel of β-clamp and blocks its loading onto DNA, which represents a new inhibitory mechanism against β-clamp function. Interestingly, the key residues responsible for this interaction on the β-clamp are well conserved among bacteria. We therefore demonstrate that Gp168 is potentially a cross-species β-clamp inhibitor, as it forms complex with the Bacillus subtilis β-clamp. Our findings reveal an alternative mechanism for bacteriophages to inhibit β-clamp and provide a new strategy to combat bacterial drug resistance.
History
DepositionMay 21, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.463
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.463
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7evp
  • Surface level: 0.463
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31339.png

Downloads & links

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Map

FileDownload / File: emd_31339.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the Gp168-beta-clamp complex
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.463 / Movie #1: 0.463
Minimum - Maximum-2.414452 - 3.8218613
Average (Standard dev.)0.007948583 (±0.09709998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 209.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z209.920209.920209.920
α/β/γ90.00090.00090.000
start NX/NY/NZ535455
NX/NY/NZ134138134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.4143.8220.008

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Supplemental data

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Sample components

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Entire : Gp168-beta-clamp complex

EntireName: Gp168-beta-clamp complex
Components
  • Complex: Gp168-beta-clamp complex
    • Complex: Beta sliding clamp 2
      • Protein or peptide: Beta sliding clamp
    • Complex: Gp168
      • Protein or peptide: Sliding clamp inhibitor

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Supramolecule #1: Gp168-beta-clamp complex

SupramoleculeName: Gp168-beta-clamp complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 100 KDa

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Supramolecule #2: Beta sliding clamp 2

SupramoleculeName: Beta sliding clamp 2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Staphylococcus aureus (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Supramolecule #3: Gp168

SupramoleculeName: Gp168 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Staphylococcus virus Twort
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Macromolecule #1: Beta sliding clamp

MacromoleculeName: Beta sliding clamp / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 41.955414 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MMEFTIKRDY FITQLNDTLK AISPRTTLPI LTGIKIDAKE HEVILTGSDS EISIEITIPK TVDGEDIVNI SETGSVVLPG RFFVDIIKK LPGKDVKLST NEQFQTLITS GHSEFNLSGL DPDQYPLLPQ VSRDDAIQLS VKVLKNVIAQ TNFAVSTSET R PVLTGVNW ...String:
MMEFTIKRDY FITQLNDTLK AISPRTTLPI LTGIKIDAKE HEVILTGSDS EISIEITIPK TVDGEDIVNI SETGSVVLPG RFFVDIIKK LPGKDVKLST NEQFQTLITS GHSEFNLSGL DPDQYPLLPQ VSRDDAIQLS VKVLKNVIAQ TNFAVSTSET R PVLTGVNW LIQENELICT ATDSHRLAVR KLQLEDVSEN KNVIIPGKAL AELNKIMSDN EEDIDIFFAS NQVLFKVGNV NF ISRLLEG HYPDTTRLFP ENYEIKLSID NGEFYHAIDR ASLLAREGGN NVIKLSTGDD VVELSSTSPE IGTVKEEVDA NDV EGGSLK ISFNSKYMMD ALKAIDNDEV EVEFFGTMKP FILKPKGDDS VTQLILPIRT Y

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Macromolecule #2: Sliding clamp inhibitor

MacromoleculeName: Sliding clamp inhibitor / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus virus Twort
Molecular weightTheoretical: 8.997979 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MLFFKEKFYN ELSYYRGGHK DLESMFELAL EYIEKLEEED EQQVTDYENA MEEELRDAVD VIESQLEIIK DIVR

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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

Concentration0.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 56.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 218035

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