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- PDB-9dls: Vibrio cholerae DnaB -

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Basic information

Entry
Database: PDB / ID: 9dls
TitleVibrio cholerae DnaB
Components
  • Replicative DNA helicase
  • ssDNA
KeywordsDNA BINDING PROTEIN/DNA / Replicative Helicase / DNA Replication / Vibrio cholerae / Helicase Loading / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


primosome complex / DNA 5'-3' helicase / DNA replication, synthesis of primer / isomerase activity / single-stranded 3'-5' DNA helicase activity / double-stranded DNA helicase activity / forked DNA-dependent helicase activity / four-way junction helicase activity / ATP hydrolysis activity / DNA binding ...primosome complex / DNA 5'-3' helicase / DNA replication, synthesis of primer / isomerase activity / single-stranded 3'-5' DNA helicase activity / double-stranded DNA helicase activity / forked DNA-dependent helicase activity / four-way junction helicase activity / ATP hydrolysis activity / DNA binding / ATP binding / metal ion 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Replicative DNA helicase
Similarity search - Component
Biological speciesVibrio cholerae (bacteria)
Vibrio cholerae O1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsMazzoletti, D. / Peng, A. / Gao, N. / Olinares, P.D.B. / Morrone, C. / Garavaglia, A. / Mendoza, A. / Chowdhury, A. / Gouda, N. / Tsoy, S. ...Mazzoletti, D. / Peng, A. / Gao, N. / Olinares, P.D.B. / Morrone, C. / Garavaglia, A. / Mendoza, A. / Chowdhury, A. / Gouda, N. / Tsoy, S. / Bhavsar, H. / Cerullo, A. / Rossi, F. / Rizzi, M. / Chait, B.T. / Miggiano, R. / Jeruzalmi, D.
Funding support United States, Italy, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1818255 United States
Fondazione CARIPLO2020_3589 Italy
Italian Ministry of Education2022LCN738 Italy
CitationJournal: Nucleic Acids Res / Year: 2025
Title: DnaB and DciA: mechanisms of helicase loading and translocation on ssDNA.
Authors: Nicholas Gao / Daniele Mazzoletti / Adele Peng / Paul Dominic B Olinares / Castrese Morrone / Andrea Garavaglia / Nourelhoda Gouda / Sergey Tsoy / Albert Mendoza / Ahammad Chowdhury / ...Authors: Nicholas Gao / Daniele Mazzoletti / Adele Peng / Paul Dominic B Olinares / Castrese Morrone / Andrea Garavaglia / Nourelhoda Gouda / Sergey Tsoy / Albert Mendoza / Ahammad Chowdhury / Antonio Cerullo / Hrutvik Bhavsar / Franca Rossi / Menico Rizzi / Brian T Chait / Riccardo Miggiano / David Jeruzalmi /
Abstract: Replicative helicases are assembled on chromosomes by helicase loaders before the initiation of DNA replication. Here, we investigate the mechanisms employed by the bacterial Vibrio cholerae (Vc) ...Replicative helicases are assembled on chromosomes by helicase loaders before the initiation of DNA replication. Here, we investigate the mechanisms employed by the bacterial Vibrio cholerae (Vc) DnaB replicative helicase and the DciA helicase loader. Structural analysis of the ATPγS form of the VcDnaB-ssDNA complex reveals a configuration distinct from that observed with GDP•AlF4. With ATPγS, the amino-terminal domain (NTD) tier, previously found as an open spiral in the GDP•AlF4 complex, adopts a closed planar arrangement. Furthermore, the DnaB subunit at the top of the carboxy-terminal domain (CTD) spiral is displaced by approximately 25 Å between the two forms. We suggest that remodeling the NTD layer between closed planar and open spiral configurations, along with the migration of two distinct CTDs to the top of the DnaB spiral, repeated three times, mediates hand-over-hand translocation. Biochemical analysis indicates that VcDciA utilizes its Lasso domain to interact with DnaB near its Docking-Helix Linker-Helix interface. Up to three copies of VcDciA bind to VcDnaB, suppressing its ATPase activity during loading onto physiological DNA substrates. Our data suggest that DciA loads DnaB onto DNA using the ring-opening mechanism.
History
DepositionSep 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 16, 2025Provider: 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: ssDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)322,68519
Polymers319,4007
Non-polymers3,28512
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Replicative DNA helicase


Mass: 52108.773 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Strain: O1
Gene: dnaB, BC353_11625, CGT77_16630, D6U24_18545, ERS013165_03687, ERS013200_03939, F0M16_21215, FLM02_14585, FLM12_17840, QXB71_003857
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A085R2T8, DNA 5'-3' helicase
#2: DNA chain ssDNA


Mass: 6747.464 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The sample sequence is different than the sequence from the coordinates because the resolution of the map was not enough to unambiguosly identify nucleobases during 3D reconstruction
Source: (synth.) Vibrio cholerae O1 (bacteria)
#3: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Hexameric DNA replicative helicase Vc-DnaB in complex with ssDNA, ATPgammaS and Mg2+COMPLEX#1-#20MULTIPLE SOURCES
2helicase Vc-DnaBCOMPLEX#11RECOMBINANT
3ssDNACOMPLEX#21MULTIPLE SOURCESThe DNA sequence of the substrate included in the sample preparation for cryo-EM is: GTC ATT AAA TAT ATA TAA AGA TCT ATA TAG AGA TCT TTT TAT TAG ATC TAC TAT TAA GGA The ssDNA sequence of the strand modelled in the cryo-EM structure is: TCC AGA TAC ACA AAA AAA AAA A
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Vibrio cholerae O1 (bacteria)127906
33Vibrio cholerae O1 (bacteria)127906
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid1
2200 mMsodium chlorideNaCl1
35 mMmagnesium chlorideMgCl21
45 mM2-MercaptoethanolHOCH2CH2SH1
55 mMATPgammaS1
SpecimenConc.: 5.62 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse and contained also the helicase loader Vc-DciA at 18 uM.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77.36 K / Temperature (min): 77.36 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 51.37 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12265
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2Leginonimage acquisition
4cryoSPARC3.3.2CTF correction
7MOLREP11.9.02model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
11cryoSPARC3.3.2classification
12cryoSPARC3.3.23D reconstruction
13PHENIX1.21.1-5286-0000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4315534
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 551305 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 73.05 / Protocol: OTHER / Space: REAL
Atomic model buildingAccession code: AF-C3LRA3-F1-model_v4 / Chain residue range: 1-468 / Source name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.37 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221645
ELECTRON MICROSCOPYf_angle_d0.38229408
ELECTRON MICROSCOPYf_dihedral_angle_d11.6638429
ELECTRON MICROSCOPYf_chiral_restr0.0373361
ELECTRON MICROSCOPYf_plane_restr0.0033772

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