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- PDB-8q72: E. coli plasmid-borne JetABCD(E248A) core in a cleavage-competent... -

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Basic information

Entry
Database: PDB / ID: 8q72
TitleE. coli plasmid-borne JetABCD(E248A) core in a cleavage-competent state
Components
  • Circular plasmid DNA (1840-MER)
  • JetA
  • JetB
  • JetC
  • JetD(E248A)
KeywordsDNA BINDING PROTEIN / SMC complexes / DNA loop extrusion / nuclease / bacterial immunity / defense system / topoisomerase / plamid restriction / MksBEFG / Wadjet / EptABCD / horizontal gene transfer / DNA kinking
Function / homology
Function and homology information


P-loop containing region of AAA domain / Uncharacterised conserved protein UCP028408 / Wadjet protein JetD, C-terminal / Domain of unknown function DUF3322 / Wadjet protein JetD, C-terminal / Uncharacterized protein conserved in bacteria N-term (DUF3322) / Protein of unknown function DUF3375 / Protein of unknown function (DUF3375) / SbcC/RAD50-like, Walker B motif / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / DUF3322 and DUF2220 domain-containing protein / DUF3375 family protein / Chromosome partition protein Smc
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.17 Å
AuthorsRoisne-Hamelin, F. / Li, Y. / Gruber, S.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)724482European Union
CitationJournal: Mol Cell / Year: 2024
Title: Structural basis for plasmid restriction by SMC JET nuclease.
Authors: Florian Roisné-Hamelin / Hon Wing Liu / Michael Taschner / Yan Li / Stephan Gruber /
Abstract: DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the ...DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.
History
DepositionAug 15, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 20, 2024Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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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
F: JetC
G: JetC
H: JetB
I: JetB
J: JetA
M: JetD(E248A)
N: JetD(E248A)
P: Circular plasmid DNA (1840-MER)
Q: Circular plasmid DNA (1840-MER)
R: Circular plasmid DNA (1840-MER)
S: Circular plasmid DNA (1840-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)864,76920
Polymers863,06016
Non-polymers1,7094
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 4 types, 12 molecules ABFGCDHIEJMN

#1: Protein
JetC


Mass: 124562.938 Da / Num. of mol.: 4 / Mutation: "G" as been added to the C-terminus.
Source method: isolated from a genetically manipulated source
Details: The last "G" in the theorical sequence is the result of a DNA cloning scar.
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: GF4-3 / Gene: GP975_00960 / Production host: Escherichia coli (E. coli) / Strain (production host): Escherichia coli (BL21) / References: UniProt: A0A6D0I2P0
#2: Protein
JetB


Mass: 28020.416 Da / Num. of mol.: 4 / Mutation: "G" as been added to the C-terminus
Source method: isolated from a genetically manipulated source
Details: The last "G" in the theorical sequence is the result of a DNA cloning scar.
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: GF4-3 / Gene: JetB / Production host: Escherichia coli (E. coli) / Strain (production host): Escherichia coli (BL21)
#3: Protein JetA


Mass: 57818.910 Da / Num. of mol.: 2
Mutation: "GPAA" has been added to the N-terminus and "G" to the C-terminus
Source method: isolated from a genetically manipulated source
Details: "GPAA" at the begining of the theorical sequence is the remaining of the purification tag after tag cleavage. The last "G" in the theorical sequence is the result of a DNA cloning scar.
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: GF4-3 / Gene: GP975_00950 / Production host: Escherichia coli (E. coli) / Strain (production host): Escherichia coli (BL21) / References: UniProt: A0A4V3QHV5
#4: Protein JetD(E248A)


Mass: 43938.492 Da / Num. of mol.: 2
Mutation: "E248A" mutation has been introduced, "GSLEVLFQ" has been added to the C-terminus.
Source method: isolated from a genetically manipulated source
Details: "GSLEVLFQ" at the C-terminus in the theorical sequence is the remaining of the tag after tag cleavage. The sequence carries the "E248A" mutation (nuclease dead).
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: GP975_00965 / Production host: Escherichia coli (E. coli) / Strain (production host): Escherichia coli (BL21) / References: UniProt: A0A3T6B0Z0

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DNA chain / Non-polymers , 2 types, 8 molecules PQRS

#5: DNA chain
Circular plasmid DNA (1840-MER)


Mass: 12303.033 Da / Num. of mol.: 4 / Mutation: The DNA was modelled as polyAT track.
Source method: isolated from a genetically manipulated source
Details: Only a portion of the plasmid (pSG6085, 1840 bp) has been modelled as polyAT track, because the complex is expected to bind in random position to the DNA and the resolution of DNA do not ...Details: Only a portion of the plasmid (pSG6085, 1840 bp) has been modelled as polyAT track, because the complex is expected to bind in random position to the DNA and the resolution of DNA do not allow any sequence assignement.
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / Strain (production host): Escherichia coli (DH5alpha)
#6: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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: JetABCD(E248A) loaded onto plasmid DNA / Type: COMPLEX
Details: E. coli JetABC, JetD(E248A) and plasmid DNA were mixed and incubated with ATP prior freezing.
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: GF4-3
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Escherichia coli (BL21)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / 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 FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 37902

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3particle selection
2EPUimage acquisition
4cryoSPARC4.1CTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
9UCSF ChimeraXmodel fitting
11PHENIX1.20.1_4487:model refinement
15cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 235956 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeInitial refinement model-IDSource nameTypeDetails
18AS8

8as8

8AS81PDBexperimental model
2AlphaFoldin silico model
3Otherin silico modelIdealized B-form DNA was flexibly fitted
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00338934
ELECTRON MICROSCOPYf_angle_d0.55153378
ELECTRON MICROSCOPYf_dihedral_angle_d19.4856352
ELECTRON MICROSCOPYf_chiral_restr0.0375966
ELECTRON MICROSCOPYf_plane_restr0.0046412

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