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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 / homologyDNA helicase, DnaB-like, C-terminal / IstB-like ATP binding protein / Superfamily 4 helicase domain profile. / IstB-like ATP-binding protein / AAA+ ATPase domain / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase C terminal domain / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / P-loop containing nucleoside triphosphate hydrolase ...DNA helicase, DnaB-like, C-terminal / IstB-like ATP binding protein / Superfamily 4 helicase domain profile. / IstB-like ATP-binding protein / AAA+ ATPase domain / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase C terminal domain / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / P-loop containing nucleoside triphosphate hydrolase / DNA replication protein DnaC/insertion sequence putative ATP-binding protein / DNA helicase, DnaB-like, N-terminal domain superfamily / DnaB-like helicase N terminal domain / primosome complex / DNA replication, synthesis of RNA primer / DNA helicase activity / DNA helicase / DNA binding / ATP binding / Replicative DNA helicase / DNA replication protein dnaC
Function and homology information
Specimen sourceEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsArias-Palomo, E. / Puri, N. / O'Shea Murray, V.L. / Yan, Q. / Berger, J.M.
Funding supportUnited States , Spain , 3 items
OrganizationGrant numberCountry
R37-071747United States
BFU2017-89143-PSpain
RYC-2015-19059Spain
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 8, 2019 / Release: Mar 6, 2019

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Structure visualization

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  • EMDB-4537
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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
Polyers482,54512
Non-polymers5,74324
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)70500
ΔGint (kcal/M)-470
Surface area (Å2)180200
MethodPISA

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Components

#1: Protein/peptide
Replicative DNA helicase


Mass: 52450.945 Da / Num. of mol.: 6 / Source: (gene. exp.) Escherichia coli (E. coli) / Gene: A1UM_04839 / Production host: Escherichia coli (E. coli) / References: UniProt: E3PC72, DNA helicase
#2: Protein/peptide
DNA replication protein dnaC /


Mass: 27973.152 Da / Num. of mol.: 6 / Source: (gene. exp.) Escherichia coli (E. coli) / Gene: A1UM_00191 / Production host: Escherichia coli (E. coli) / References: UniProt: L3QJA3
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 7 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Formula: Mg / Magnesium
#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 / Formula: C10H14BeF3N5O10P2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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 / 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
MicroscopyMicroscope model: 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
phenix.real_space_refine1.13_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 selectionNumber of particles selected: 551641
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 104913 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL
RefineStereochemistry 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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