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- PDB-6qel: E. coli DnaBC apo complex -

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Basic information

Entry
Database: PDB / ID: 6qel
TitleE. coli DnaBC apo complex
Components
  • DNA replication protein dnaC
  • Replicative DNA helicase
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 / DNA helicase complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / replisome / DNA replication, synthesis of primer / DNA strand elongation involved in DNA replication ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DNA helicase complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / replisome / DNA replication, synthesis of primer / DNA strand elongation involved in DNA replication / 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 helicase / DNA replication / hydrolase activity / 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 / Replicative DNA helicase / DNA replication protein dnaC / Replicative DNA helicase / DNA replication protein DnaC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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
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
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

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-4537
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)488,28836
Polymers482,54512
Non-polymers5,74324
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area70500 Å2
ΔGint-470 kcal/mol
Surface area180200 Å2
MethodPISA

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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 (E. coli) / Gene: A1UM_04839 / Production host: Escherichia coli (E. coli)
References: UniProt: E3PC72, UniProt: P0ACB0*PLUS, 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: A1UM_00191 / Production host: Escherichia coli (E. coli) / References: UniProt: L3QJA3, UniProt: P0AEF0*PLUS
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
#5: 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

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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 DnaBC apo complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.48 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grids where treated with poly-lys prior to use / 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 FIELDBright-field microscopy / Nominal defocus max: 2700 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 61 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: 551641
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104913 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008265416
ELECTRON MICROSCOPYf_angle_d0.9761118245
ELECTRON MICROSCOPYf_chiral_restr0.05294984
ELECTRON MICROSCOPYf_plane_restr0.00449755
ELECTRON MICROSCOPYf_dihedral_angle_d13.036825959

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