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- PDB-6qem: E. coli DnaBC complex bound to ssDNA -

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Basic information

Entry
Database: PDB / ID: 6qem
TitleE. coli DnaBC complex bound to ssDNA
Components
  • DNA replication protein DnaC
  • Replicative DNA helicase
  • ssDNA
KeywordsREPLICATION / Helicase / helicase loader / AAA+ / RecA
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 / DNA strand elongation involved in DNA replication / 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 replication protein DnaC/insertion sequence putative ATP-binding protein / IstB-like ATP-binding protein / IstB-like ATP binding protein / DNAb Helicase; Chain A / DNAb Helicase; Chain A / 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 ...DNA replication protein DnaC/insertion sequence putative ATP-binding protein / IstB-like ATP-binding protein / IstB-like ATP binding protein / DNAb Helicase; Chain A / DNAb Helicase; Chain A / 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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Chem-08T / ADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / Replicative DNA helicase / DNA replication protein DnaC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsArias-Palomo, E. / Puri, N. / O'Shea Murray, V.L. / Yan, Q. / Berger, J.M.
Funding support United States, Spain, 3items
OrganizationGrant numberCountry
R37-071747 United States
BFU2017-89143-P Spain
RYC-2015-19059 Spain
CitationJournal: Mol Cell / Year: 2019
Title: Physical Basis for the Loading of a Bacterial Replicative Helicase onto DNA.
Authors: Ernesto Arias-Palomo / Neha Puri / Valerie L O'Shea Murray / Qianyun Yan / James M Berger /
Abstract: In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we ...In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å-resolution structures of the E. coli DnaB⋅DnaC helicase⋅loader complex with nucleotide in pre- and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition.
History
DepositionJan 8, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2019Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: software / Item: _software.name
Revision 1.3Dec 4, 2019Group: Derived calculations / Category: struct_conf / struct_sheet / struct_sheet_range
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.5May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-4538
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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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 replication protein DnaC
H: DNA replication protein DnaC
I: DNA replication protein DnaC
J: DNA replication protein DnaC
K: DNA replication protein DnaC
L: DNA replication protein DnaC
M: ssDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)498,59629
Polymers493,45113
Non-polymers5,14616
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area75480 Å2
ΔGint-409 kcal/mol
Surface area174470 Å2
MethodPISA

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Components

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Protein , 2 types, 12 molecules ABCDEFGHIJKL

#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 (E. coli) / Gene: dnaB, groP, grpA, b4052, JW4012 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ACB0, DNA helicase
#2: Protein
DNA replication protein DnaC


Mass: 27973.152 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: dnaC, dnaD, b4361, JW4325 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AEF0, DNA helicase

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DNA chain , 1 types, 1 molecules M

#3: DNA chain ssDNA


Mass: 10905.983 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 3 types, 16 molecules

#4: Chemical
ChemComp-08T / [[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-oxidanyl-phosphoryl]oxy-tris(fluoranyl)beryllium


Mass: 492.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H14BeF3N5O10P2
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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

Component
IDNameTypeEntity IDParent-IDSource
1E. coli DnaBC complex bound to ssDNACOMPLEX#1-#30MULTIPLE SOURCES
2DnaBCCOMPLEX#1-#21RECOMBINANT
3ssDNACOMPLEX#31RECOMBINANT
Molecular weightValue: 0.49 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grids were pre-treated with poly-lys / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2700 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 58 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
PHENIX1.13_2998refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
5RELION2.1CTF correction
10PHENIX1.13model refinement
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 473888
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229097 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009666399
ELECTRON MICROSCOPYf_angle_d1.0537120027
ELECTRON MICROSCOPYf_chiral_restr0.0555092
ELECTRON MICROSCOPYf_plane_restr0.0059827
ELECTRON MICROSCOPYf_dihedral_angle_d13.259426314

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