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- PDB-9qxt: E. coli JetABC dimer in the DNA boarding-holding state -

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Open data


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Basic information

Entry
Database: PDB / ID: 9qxt
TitleE. coli JetABC dimer in the DNA boarding-holding state
Components
  • Circular plasmid DNA (1843-MER)
  • JetA
  • JetB
  • JetC
KeywordsDNA BINDING PROTEIN / SMC complexes / Wadjet / JetABCD / DNA loop extrusion
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.13 Å
AuthorsRoisne-Hamelin, F. / Gruber, S.
Funding supportEuropean Union, Switzerland, 2items
OrganizationGrant numberCountry
European Research Council (ERC)724482European Union
Swiss National Science Foundation10001017 Switzerland
CitationJournal: Mol Cell / Year: 2025
Title: Mechanism of DNA entrapment by a loop-extruding Wadjet SMC motor.
Authors: Florian Roisné-Hamelin / Hon Wing Liu / Nils Maréchal / Emiko Uchikawa / Alexandre Durand / Stephan Gruber /
Abstract: Structural maintenance of chromosome (SMC) complexes perform critical functions by folding DNA through loop extrusion. The choreography and outcome of SMC DNA loading prior to loop extrusion, ...Structural maintenance of chromosome (SMC) complexes perform critical functions by folding DNA through loop extrusion. The choreography and outcome of SMC DNA loading prior to loop extrusion, however, remain elusive. Here, we use cryo-electron microscopy to determine structures of the prokaryotic SMC Wadjet undergoing DNA loading. We show that an initial ATP-triggered relocation of both SMC dimers exposes a DNA-binding pocket and aligns two opened motor units on a DNA double helix. Subsequent ATP hydrolysis drives a nearly 360° rotation of each SMC dimer, closing the motor units around DNA in a sequential manner. This process leads to a DNA-holding conformation-an anticipated key intermediate in loop extrusion-with the DNA held within the kleisin/KITE sub-compartment. Our findings elucidate the mechanism of topological DNA loading by an SMC motor, revealing a straight DNA double helix with motor units oriented tail-to-tail in DNA-holding conformations as the likely starting point of DNA loop extrusion.
History
DepositionApr 16, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: JetB
D: JetB
E: JetA
A: JetC
B: JetC
H: JetB
I: JetB
J: JetA
P: Circular plasmid DNA (1843-MER)
Q: Circular plasmid DNA (1843-MER)
F: JetC
G: JetC


Theoretical massNumber of molelcules
Total (without water)762,82812
Polymers762,82812
Non-polymers00
Water00
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

#1: Protein
JetB


Mass: 28004.350 Da / Num. of mol.: 4 / Mutation: C19S
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: ECIG_04711 / Production host: Escherichia coli BL21 (bacteria)
#2: Protein JetA


Mass: 57786.848 Da / Num. of mol.: 2 / Mutation: C36A
Source method: isolated from a genetically manipulated source
Details: The last "G" in the theorical sequence is the result of a DNA cloning scar. "GPAA" at the begining of the theorical sequence is the remaining of the purification tag after tag cleavage. The ...Details: The last "G" in the theorical sequence is the result of a DNA cloning scar. "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, GQA06_02520 / Production host: Escherichia coli BL21 (bacteria)
#3: Protein
JetC


Mass: 124570.797 Da / Num. of mol.: 4 / Mutation: C61L,C336H,C401R,C568A,N721C,C753S,C942S,C1006S
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: C9160_20650 / Production host: Escherichia coli BL21 (bacteria)
#4: DNA chain Circular plasmid DNA (1843-MER)


Mass: 18477.025 Da / Num. of mol.: 2 / Mutation: The DNA was modelled as polyAT track.
Source method: isolated from a genetically manipulated source
Details: Only a portion of the plasmid (pSG6085, 1843 bp) has been modelled as polyAT track, because the complex is expected to load at random positions and the local resolution of the DNA does not ...Details: Only a portion of the plasmid (pSG6085, 1843 bp) has been modelled as polyAT track, because the complex is expected to load at random positions and the local resolution of the DNA does not allow any sequence assignment.
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / Strain (production host): pirHC
Has protein modificationN

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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: Middle-crosslinked JetABC loading on plasmid DNA / Type: COMPLEX
Details: E. coli cysteine-less JetABC crosslinked at the "middle" position was incubated with plasmid DNA in presence of ATP prior grid freezing.
Entity ID: all / 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
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 FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 26586

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Processing

EM software
IDNameVersionCategory
12cryoSPARCV4.63D reconstruction
13PHENIX1.21.2_5419:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63069 / 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
28bfn

8bfn

8bfn2PDBexperimental model
38q72

8q72

8q723PDBexperimental model
4Otherin silico modelIdealized B-form DNA was flexibly fitted
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00541608
ELECTRON MICROSCOPYf_angle_d0.62556790
ELECTRON MICROSCOPYf_dihedral_angle_d15.156692
ELECTRON MICROSCOPYf_chiral_restr0.0396384
ELECTRON MICROSCOPYf_plane_restr0.0057056

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