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- EMDB-46984: Vibrio cholerae DnaB -

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Basic information

Entry
Database: EMDB / ID: EMD-46984
TitleVibrio cholerae DnaB
Map dataLow resolution map from heterogeneus refinement featuring ssDNA extension.
Sample
  • Complex: Hexameric DNA replicative helicase Vc-DnaB in complex with ssDNA, ATPgammaS and Mg2+
    • Complex: helicase Vc-DnaB
      • Protein or peptide: Replicative DNA helicase
    • Complex: ssDNA
      • DNA: ssDNA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsReplicative 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
Replicative DNA helicase
Similarity search - Component
Biological speciesVibrio cholerae O1 (bacteria) / Vibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsMazzoletti D / Peng A / Gao N / Olinares PDB / Morrone C / Garavaglia A / Mendoza A / Chowdhury A / Gouda N / Tsoy S ...Mazzoletti D / Peng A / Gao N / Olinares PDB / Morrone C / Garavaglia A / Mendoza A / Chowdhury A / Gouda N / Tsoy S / Bhavsar H / Cerullo A / Rossi F / Rizzi M / Chait BT / Miggiano R / Jeruzalmi D
Funding support United States, Italy, 3 items
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, 2024-
Header (metadata) releaseJul 16, 2025-
Map releaseJul 16, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46984.png

Downloads & links

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Map

FileDownload / File: emd_46984.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLow resolution map from heterogeneus refinement featuring ssDNA extension.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 360 pix.
= 389.736 Å		1.08 Å/pix.
x 360 pix.
= 389.736 Å		1.08 Å/pix.
x 360 pix.
= 389.736 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.207
Minimum - Maximum-0.0017811931 - 2.0141857
Average (Standard dev.)0.0007216623 (±0.018786848)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 389.736 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Low resolution map from heterogeneus refinement featuring ssDNA...

Fileemd_46984_additional_1.map
AnnotationLow resolution map from heterogeneus refinement featuring ssDNA extension.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Low resolution map from heterogeneus refinement featuring ssDNA...

Fileemd_46984_half_map_1.map
AnnotationLow resolution map from heterogeneus refinement featuring ssDNA extension.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Low resolution map from heterogeneus refinement featuring ssDNA...

Fileemd_46984_half_map_2.map
AnnotationLow resolution map from heterogeneus refinement featuring ssDNA extension.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexameric DNA replicative helicase Vc-DnaB in complex with ssDNA,...

EntireName: Hexameric DNA replicative helicase Vc-DnaB in complex with ssDNA, ATPgammaS and Mg2+
Components
  • Complex: Hexameric DNA replicative helicase Vc-DnaB in complex with ssDNA, ATPgammaS and Mg2+
    • Complex: helicase Vc-DnaB
      • Protein or peptide: Replicative DNA helicase
    • Complex: ssDNA
      • DNA: ssDNA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Hexameric DNA replicative helicase Vc-DnaB in complex with ssDNA,...

SupramoleculeName: Hexameric DNA replicative helicase Vc-DnaB in complex with ssDNA, ATPgammaS and Mg2+
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: helicase Vc-DnaB

SupramoleculeName: helicase Vc-DnaB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Vibrio cholerae O1 (bacteria)

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Supramolecule #3: ssDNA

SupramoleculeName: ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: The 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 ...Details: The 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
Source (natural)Organism: Vibrio cholerae O1 (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 5'-3' helicase
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: O1
Molecular weightTheoretical: 52.108773 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MADPRTDNRN RKIPDAQVDA IKVPPHSLEA EQSVIGGLLL DNERWDTVSE HVMTQDFYSR PHRLIFDGVK SILEAGKPLD LITLSEYLE QREQLEDVGG FAYLADLAKN TPSAANINAY AEIVAERALV RNLIGVANEI ADAGYDPQGR NAEDLLDLAE S KVFAIAEA ...String:
MADPRTDNRN RKIPDAQVDA IKVPPHSLEA EQSVIGGLLL DNERWDTVSE HVMTQDFYSR PHRLIFDGVK SILEAGKPLD LITLSEYLE QREQLEDVGG FAYLADLAKN TPSAANINAY AEIVAERALV RNLIGVANEI ADAGYDPQGR NAEDLLDLAE S KVFAIAEA RTSENEGPKN VDSILERTLE RIELLYKTPQ DGVTGVNTGF TDLNKKTAGL QGSDLIIVAA RPSMGKTTFA MN LCENAAM EQDKPVLIFS LEMPAEQIMM RMLASLSRVD QTKIRTGQLD DEDWARISST MGILMEKKNM YIDDSSGLTP TEV RSRARR IAREHGGLSL IMVDYLQLMR VPALTDNRTL EIAEISRSLK ALAKELNVPV VALSQLNRSL EQRADKRPVN SDLR ESGSI EQDADLIMFI YRDEVYHPDS PLKGTAEIII GKQRNGPIGS VRLTFQGHYS RFDNYAGPAF DDE

UniProtKB: Replicative DNA helicase

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Macromolecule #2: ssDNA

MacromoleculeName: ssDNA / type: dna / ID: 2
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
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Vibrio cholerae O1 (bacteria)
Molecular weightTheoretical: 6.747464 KDa
SequenceString:
(DT)(DC)(DC)(DA)(DG)(DA)(DT)(DA)(DC)(DA) (DC)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / 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 state3D array

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Sample preparation

Concentration5.62 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mM4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid
200.0 mMsodium chlorideNaCl
5.0 mMmagnesium chlorideMgCl2
5.0 mM2-MercaptoethanolHOCH2CH2SH
5.0 mMATPgammaS
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was monodisperse and contained also the helicase loader Vc-DciA at 18 uM.

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 77.36 K / Max: 77.36 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 12265 / Average exposure time: 2.0 sec. / Average electron dose: 51.37 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4315534
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7t20

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 551305
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 200 / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

l_v4


Chain - Chain ID: A / Chain - Residue range: 1-468 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 73.05
Output model

PDB-9dls:
Vibrio cholerae DnaB

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