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- PDB-8as8: E. coli Wadjet JetABC monomer -

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Basic information

Entry
Database: PDB / ID: 8as8
TitleE. coli Wadjet JetABC monomer
Components
  • JetA
  • JetB
  • JetC
KeywordsDNA BINDING PROTEIN / SMC complexes / bacterial immunity / defense system / DNA loop extrusion / Wadjet / JetABCD / DNA cleavage / plasmid restriction / mksBEFG / mobile genetic elements / horizontal gene transfer / eptABCD / mukBEF
Function / homologyWadjet protein JetB / Domain of unknown function (DUF4194) / Protein of unknown function DUF3375 / Protein of unknown function (DUF3375) / P-loop containing nucleoside triphosphate hydrolase / ADENOSINE-5'-DIPHOSPHATE / DUF4194 domain-containing protein / ATP synthase / DUF3375 family protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsRoisne-Hamelin, F. / Beckert, B. / Li, Y. / Myasnikov, A. / Gruber, S.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)724482European Union
CitationJournal: Mol Cell / Year: 2022
Title: DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage.
Authors: Hon Wing Liu / Florian Roisné-Hamelin / Bertrand Beckert / Yan Li / Alexander Myasnikov / Stephan Gruber /
Abstract: Structural maintenance of chromosome (SMC) complexes fold DNA by loop extrusion to support chromosome segregation and genome maintenance. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC ...Structural maintenance of chromosome (SMC) complexes fold DNA by loop extrusion to support chromosome segregation and genome maintenance. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with roles in bacterial immunity against selfish DNA. Here, we show that JetABCD restricts circular plasmids with an upper size limit of about 100 kb, whereas a linear plasmid evades restriction. Purified JetABCD complexes cleave circular DNA molecules, regardless of the DNA helical topology; cleavage is DNA sequence nonspecific and depends on the SMC ATPase. A cryo-EM structure reveals a distinct JetABC dimer-of-dimers geometry, with the two SMC dimers facing in opposite direction-rather than the same as observed with MukBEF. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and propose the "total extrusion model" for DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a JetABCD complex. Total extrusion cannot be achieved on the larger chromosome, explaining how self-DNA may evade processing.
History
DepositionAug 18, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 28, 2022Group: Data processing / Database references / Category: citation / em_3d_reconstruction
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_PubMed / _em_3d_reconstruction.resolution
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: JetC
B: JetC
C: JetB
D: JetB
E: JetA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)369,3727
Polymers368,5185
Non-polymers8542
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein JetC


Mass: 124562.938 Da / Num. of mol.: 2 / Mutation: G added to C-terminus
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: C9160_20650 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A4T5T6V2
#2: Protein JetB


Mass: 28020.416 Da / Num. of mol.: 2 / Mutation: G added to C-terminus
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: BvCmsKKP036_00455 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A4C9B499
#3: Protein JetA


Mass: 63350.883 Da / Num. of mol.: 1
Mutation: MAHHHHHHHHHHGGSSAWSHPQFEKGGGSGGGSGGGSWSHPQFEKLEVLFQGPAA tag added at N-terminus; G added to C-terminus
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: GF4-3 / Gene: WP_171460373 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A4V3QHV5
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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: JetABC(D) / Type: COMPLEX
Details: E. coli JetABCD was purified by gel filtration. Note that JetD is not visible in the map.
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: GF4-3
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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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 defocus max: 1500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: RELION / Version: 4 / Category: classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124689 / Symmetry type: POINT

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