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- PDB-9eco: E. coli DnaB bound to three DnaG C-terminal domains, ssDNA, ADP a... -

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Basic information

Entry
Database: PDB / ID: 9eco
TitleE. coli DnaB bound to three DnaG C-terminal domains, ssDNA, ADP and AlF4
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • DNA primase
  • Replicative DNA helicase
KeywordsDNA BINDING PROTEIN/DNA / helicase / SF4 / AAA+ / REPLICATION-DNA complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DNA primase DnaG / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / DNA replication, synthesis of primer ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DNA primase DnaG / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / DNA replication, synthesis of primer / replisome / response to ionizing radiation / replication fork processing / DNA replication initiation / DNA-directed RNA polymerase complex / DNA helicase activity / helicase activity / DNA-directed RNA polymerase activity / 5'-3' DNA helicase activity / DNA replication / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
DNA primase DnaG, DnaB-binding domain / DNA primase DnaG DnaB-binding / DNA primase DnaG DnaB-binding / DNA primase, DnaB-helicase binding domain / DnaB-helicase binding domain of primase / Zinc finger, CHC2-type / DNA primase, DnaG / DNA primase, catalytic core, N-terminal / DNA primase DnaG, bacteria / Bacterial DnaG primase, TOPRIM domain ...DNA primase DnaG, DnaB-binding domain / DNA primase DnaG DnaB-binding / DNA primase DnaG DnaB-binding / DNA primase, DnaB-helicase binding domain / DnaB-helicase binding domain of primase / Zinc finger, CHC2-type / DNA primase, DnaG / DNA primase, catalytic core, N-terminal / DNA primase DnaG, bacteria / Bacterial DnaG primase, TOPRIM domain / DNA primase, catalytic core, N-terminal domain superfamily / : / CHC2 zinc finger / DNA primase catalytic core, N-terminal domain / zinc finger / DNA Primase, CHC2-type zinc finger / DNA helicase, DnaB type / Toprim-like / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / TOPRIM / Toprim domain profile. / TOPRIM domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / TETRAFLUOROALUMINATE ION / DNA / DNA (> 10) / DNA primase / Replicative DNA helicase DnaB
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsOakley, A.J. / Xu, Z.Q.
Funding support Australia, 1items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP210100365 Australia
CitationJournal: To Be Published
Title: Sequence of conformation changes of DnaB helicase during DNA unwinding and priming in Escherichia coli
Authors: Oakley, A.J. / Xu, Z.Q.
History
DepositionNov 14, 2024Deposition site: RCSB / Processing site: RCSB
SupersessionDec 11, 2024ID: 7T23
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replicative DNA helicase
B: Replicative DNA helicase
C: Replicative DNA helicase
D: Replicative DNA helicase
E: Replicative DNA helicase
F: Replicative DNA helicase
G: DNA primase
H: DNA primase
M: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)356,58324
Polymers353,8109
Non-polymers2,77215
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

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Protein , 2 types, 8 molecules ABCDEFGH

#1: Protein
Replicative DNA helicase


Mass: 52406.918 Da / Num. of mol.: 6 / Mutation: F103C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: dnaB, groP, grpA, b4052, JW4012 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RECA- / References: UniProt: P0ACB0, DNA helicase
#2: Protein DNA primase


Mass: 16664.918 Da / Num. of mol.: 2 / Fragment: C-terminal domain / Mutation: R568C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: dnaG, dnaP, parB, b3066, JW3038 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RECA- / References: UniProt: P0ABS5, DNA primase DnaG

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DNA chain , 1 types, 1 molecules M

#3: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 6038.899 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 3 types, 15 molecules

#4: Chemical
ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: AlF4
#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
Has protein modificationY

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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: E. coli DnaB bound to DnaG C-terminal domain and ssDNA
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.353 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Escherichia coli K-12 (bacteria)83333
21Escherichia coli (E. coli)562K-12
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
Details: 20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 5 mM MgCl2, 3 mM DTT, 0.25 mM EDTA and 100 micromolar ADP, 0.5 mM AlCl3, 5 mM NaF.
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were Glow-discharged grids for four min at 0.39 mBar and 15 mA.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Chamber temperature: 279 K
Details: 3 microL of sample was applied to glow-discharged grids. Grids were blotted at 6 degrees C for 3.5 s with no extra blot force.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1200 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
2EPUimage acquisition
4cryoSPARC4.6.0CTF correction
7PHENIX1.19model fitting
9PHENIX1.19model refinement
10cryoSPARC4.6.0initial Euler assignment
11cryoSPARC4.6.0final Euler assignment
12cryoSPARC4.6.0classification
13cryoSPARC4.6.03D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 518624
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78705 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Correlation Coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00323861
ELECTRON MICROSCOPYf_angle_d0.49832357
ELECTRON MICROSCOPYf_dihedral_angle_d8.2463425
ELECTRON MICROSCOPYf_chiral_restr0.0373682
ELECTRON MICROSCOPYf_plane_restr0.0044236

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