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- EMDB-25609: E. coli DnaB bound to three DnaG C-terminal domains, ssDNA, ADP a... -

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Basic information

Entry
Database: EMDB / ID: EMD-25609
TitleE. coli DnaB bound to three DnaG C-terminal domains, ssDNA, ADP and AlF4
Map data
Sample
  • Complex: E. coli DnaB DnaG C-terminal domain bound to ssDNA
    • Protein or peptide: Replicative DNA helicase
    • Protein or peptide: DNA primasePrimase
    • DNA: DNA (5'-D(*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
Keywordshelicase / 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 / DNA primase DnaG / DNA helicase complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / DNA primase activity / DNA replication, synthesis of primer ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DNA primase DnaG / DNA helicase complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / DNA primase activity / DNA replication, synthesis of primer / replisome / DNA duplex unwinding / response to ionizing radiation / DNA unwinding involved in DNA replication / replication fork processing / DNA replication initiation / DNA helicase activity / DNA-directed RNA polymerase complex / helicase activity / DNA helicase / DNA replication / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / identical protein binding / cytosol / cytoplasm
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, CHC2-type zinc finger / DNA primase, catalytic core, N-terminal domain superfamily / CHC2 zinc finger / DNA primase catalytic core, N-terminal domain / zinc finger / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / Toprim-like / 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
DNA primase / Replicative DNA helicase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsOakley AJ / Xu ZQ
Funding support Australia, 1 items
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 AJ / Xu ZQ
History
DepositionDec 2, 2021-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25609.png

Downloads & links

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Map

FileDownload / File: emd_25609.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.00902
Minimum - Maximum-0.026967496 - 0.05538974
Average (Standard dev.)-0.0000071371815 (±0.0030485457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 248.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : E. coli DnaB DnaG C-terminal domain bound to ssDNA

EntireName: E. coli DnaB DnaG C-terminal domain bound to ssDNA
Components
  • Complex: E. coli DnaB DnaG C-terminal domain bound to ssDNA
    • Protein or peptide: Replicative DNA helicase
    • Protein or peptide: DNA primasePrimase
    • DNA: DNA (5'-D(*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: E. coli DnaB DnaG C-terminal domain bound to ssDNA

SupramoleculeName: E. coli DnaB DnaG C-terminal domain bound to ssDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Replicative DNA helicase

MacromoleculeName: Replicative DNA helicase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 52.406918 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGNKPFNKQ QAEPRERDPQ VAGLKVPPHS IEAEQSVLGG LMLDNERWDD VAERVVADDF YTRPHRHIFT EMARLQESGS PIDLITLAE SLERQGQLDS VGGCAYLAEL SKNTPSAANI SAYADIVRER AVVREMISVA NEIAEAGFDP QGRTSEDLLD L AESRVFKI ...String:
MAGNKPFNKQ QAEPRERDPQ VAGLKVPPHS IEAEQSVLGG LMLDNERWDD VAERVVADDF YTRPHRHIFT EMARLQESGS PIDLITLAE SLERQGQLDS VGGCAYLAEL SKNTPSAANI SAYADIVRER AVVREMISVA NEIAEAGFDP QGRTSEDLLD L AESRVFKI AESRANKDEG PKNIADVLDA TVARIEQLFQ QPHDGVTGVN TGYDDLNKKT AGLQPSDLII VAARPSMGKT TF AMNLVEN AAMLQDKPVL IFSLEMPSEQ IMMRSLASLS RVDQTKIRTG QLDDEDWARI SGTMGILLEK RNIYIDDSSG LTP TEVRSR ARRIAREHGG IGLIMIDYLQ LMRVPALSDN RTLEIAEISR SLKALAKELN VPVVALSQLN RSLEQRADKR PVNS DLRES GSIEQDADLI MFIYRDEVYH ENSDLKGIAE IIIGKQRNGP IGTVRLTFNG QWSRFDNYAG PQYDDE

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Macromolecule #2: DNA primase

MacromoleculeName: DNA primase / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO / EC number: DNA primase DnaG
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 16.664918 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
AAESGVSRPV PQLKRTTMRI LIGLLVQNPE LATLVPPLEN LDENKLPGLG LFRELVNTCL SQPGLTTGQL LEHYRGTNNA ATLEKLSMW DDIADKNIAE QTFTDSLNHM FDSLLELRQE ELIARERTHG LSNEECLELW TLNQELAKK

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Macromolecule #3: DNA (5'-D(*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*...

MacromoleculeName: DNA (5'-D(*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 6.038899 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #5: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 5 / Number of copies: 5 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 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.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE
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; ...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.
VitrificationCryogen name: ETHANE / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 657240
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.07)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.07)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.07)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.07) / Number images used: 46096

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Target criteria: Correlation Coefficient
Output model

PDB-7t22:
E. coli DnaB bound to three DnaG C-terminal domains, ssDNA, ADP and AlF4

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