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- PDB-7evp: Cryo-EM structure of the Gp168-beta-clamp complex -

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Basic information

Entry
Database: PDB / ID: 7evp
TitleCryo-EM structure of the Gp168-beta-clamp complex
Components
  • Beta sliding clamp
  • Sliding clamp inhibitor
KeywordsANTIMICROBIAL PROTEIN / gp168 / DNA beta-clamp / cross-species 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
MethodELECTRON MICROSCOPY / single 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, 1items
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, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Beta sliding clamp
C: Sliding clamp inhibitor
B: Beta sliding clamp
D: Sliding clamp inhibitor


Theoretical massNumber of molelcules
Total (without water)101,9074
Polymers101,9074
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6790 Å2
ΔGint-20 kcal/mol
Surface area36240 Å2
MethodPISA

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Components

#1: Protein Beta sliding clamp / Beta clamp / Sliding clamp / Beta-clamp processivity factor / DNA polymerase III beta sliding clamp ...Beta clamp / Sliding clamp / Beta-clamp processivity factor / DNA polymerase III beta sliding clamp subunit / DNA polymerase III subunit beta


Mass: 41955.414 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: dnaN
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A024
#2: Protein Sliding clamp inhibitor / gp168


Mass: 8997.979 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus virus Twort / Gene: TwortDSMZ_173
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A6H0X5G8

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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

Component
IDNameTypeEntity IDParent-IDSource
1Gp168-beta-clamp complexCOMPLEXall0MULTIPLE SOURCES
2Beta sliding clamp 2COMPLEX#11RECOMBINANT
3Gp168COMPLEX#21RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Staphylococcus aureus (bacteria)1280
33Staphylococcus virus Twort55510
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
33Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 56 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM softwareName: cryoSPARC / Version: 3 / Category: 3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 218035 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0086720
ELECTRON MICROSCOPYf_angle_d0.8519104
ELECTRON MICROSCOPYf_dihedral_angle_d11.3654120
ELECTRON MICROSCOPYf_chiral_restr0.061068
ELECTRON MICROSCOPYf_plane_restr0.0061188

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