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- PDB-7t20: E. coli DnaB bound to ssDNA and AMPPNP -

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Basic information

Entry
Database: PDB / ID: 7t20
TitleE. coli DnaB bound to ssDNA and AMPPNP
Components
  • DNA (5'-D(*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Replicative DNA helicase
KeywordsREPLICATION/DNA / helicase / SF4 / AAA+ / REPLICATION-DNA complex
Function / homology
Function and homology information


DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA replication, synthesis of primer / replisome / DNA duplex unwinding ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA replication, synthesis of primer / replisome / DNA duplex unwinding / response to ionizing radiation / replication fork processing / DNA unwinding involved in DNA replication / DNA replication initiation / DNA helicase activity / helicase activity / DNA replication / DNA helicase / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA helicase, DnaB type / 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. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Replicative DNA helicase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 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
DepositionDec 2, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 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

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
M: DNA (5'-D(*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)322,86715
Polymers320,7457
Non-polymers2,1228
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36290 Å2
ΔGint-238 kcal/mol
Surface area106110 Å2

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Components

#1: Protein
Replicative DNA helicase


Mass: 52450.945 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: dnaB, groP, grpA, b4052, JW4012 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RecA- / References: UniProt: P0ACB0, DNA helicase
#2: DNA chain DNA (5'-D(*TP*TP*TP*TP*TP*TP*TP*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)
#3: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 ssDNA / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli K-12 (bacteria)83333
21Escherichia coli (E. coli)562
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.
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids covered with a 2 nm continuous carbon film. Glow-discharged grids were floated on 40 microL drops of a 0.1% (w/v) aqueous solution of poly-lysine hydrobromide (MW 30-70 kDa; ...Details: Grids covered with a 2 nm continuous carbon film. Glow-discharged grids were floated on 40 microL drops of a 0.1% (w/v) aqueous solution of poly-lysine hydrobromide (MW 30-70 kDa; Polysciences, Inc.) for two min, briefly rinsed with MilliQ water 4 times and then allow to air dry.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil 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: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_4370: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.07particle selection
2EPUimage acquisition
4RELION3.07CTF correction
7PHENIX1.19model fitting
9PHENIX1.19model refinement
10RELION3.07initial Euler assignment
11RELION3.07final Euler assignment
12RELION3.07classification
13RELION3.073D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1098240
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 373620 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Correlation Coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221423
ELECTRON MICROSCOPYf_angle_d0.42829035
ELECTRON MICROSCOPYf_dihedral_angle_d11.2778203
ELECTRON MICROSCOPYf_chiral_restr0.0373298
ELECTRON MICROSCOPYf_plane_restr0.0043812

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