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- PDB-8btg: Cryo-EM structure of the bacterial replication origin opening bas... -

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Basic information

Entry
Database: PDB / ID: 8btg
TitleCryo-EM structure of the bacterial replication origin opening basal unwinding system
Components
  • Chromosomal replication initiator protein DnaA
  • DNA (41-MER)
  • DNA (5'-D(P*AP*CP*TP*TP*AP*TP*CP*CP*AP*CP*AP*AP*AP*TP*CP*CP*AP*CP*AP*GP*GP*CP*CP*C)-3')
KeywordsREPLICATION / DnaA / cryo-EM / BUS complex / replication initiation
Function / homology
Function and homology information


DNA replication origin binding / regulation of DNA replication / DNA replication initiation / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
DnaA N-terminal domain / DnaA N-terminal domain / DnaA, N-terminal domain superfamily / Chromosomal replication control, initiator DnaA / Chromosomal replication initiator, DnaA C-terminal / Chromosomal replication control, initiator DnaA, conserved site / Bacterial dnaA protein helix-turn-helix / DnaA protein signature. / Bacterial dnaA protein helix-turn-helix domain / Chromosomal replication control, initiator DnaA-like ...DnaA N-terminal domain / DnaA N-terminal domain / DnaA, N-terminal domain superfamily / Chromosomal replication control, initiator DnaA / Chromosomal replication initiator, DnaA C-terminal / Chromosomal replication control, initiator DnaA, conserved site / Bacterial dnaA protein helix-turn-helix / DnaA protein signature. / Bacterial dnaA protein helix-turn-helix domain / Chromosomal replication control, initiator DnaA-like / Chromosomal replication initiator protein DnaA / Bacterial dnaA protein / Trp repressor/replication initiator / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Chromosomal replication initiator protein DnaA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPelliciari, S. / Bodet-Lefevre, S. / Murray, H. / Ilangovan, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust204985/Z/16/Z United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: The bacterial replication origin BUS promotes nucleobase capture.
Authors: Simone Pelliciari / Salomé Bodet-Lefèvre / Stepan Fenyk / Daniel Stevens / Charles Winterhalter / Frederic D Schramm / Sara Pintar / Daniel R Burnham / George Merces / Tomas T Richardson / ...Authors: Simone Pelliciari / Salomé Bodet-Lefèvre / Stepan Fenyk / Daniel Stevens / Charles Winterhalter / Frederic D Schramm / Sara Pintar / Daniel R Burnham / George Merces / Tomas T Richardson / Yumiko Tashiro / Julia Hubbard / Hasan Yardimci / Aravindan Ilangovan / Heath Murray /
Abstract: Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is ...Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaA assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).
History
DepositionNov 28, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chromosomal replication initiator protein DnaA
B: Chromosomal replication initiator protein DnaA
C: Chromosomal replication initiator protein DnaA
D: Chromosomal replication initiator protein DnaA
E: Chromosomal replication initiator protein DnaA
F: Chromosomal replication initiator protein DnaA
G: Chromosomal replication initiator protein DnaA
X: DNA (5'-D(P*AP*CP*TP*TP*AP*TP*CP*CP*AP*CP*AP*AP*AP*TP*CP*CP*AP*CP*AP*GP*GP*CP*CP*C)-3')
Y: DNA (41-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)380,31323
Polymers376,5939
Non-polymers3,72014
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Chromosomal replication initiator protein DnaA


Mass: 50929.980 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria)
Gene: dnaA, B4417_2456, C0W65_09430, CFD21_00040, J5227_06470, NRS6116_00005, NRS6202_21170, SC09_Contig28orf00336
Production host: Escherichia coli (E. coli) / References: UniProt: A0A063XAK9
#2: DNA chain DNA (5'-D(P*AP*CP*TP*TP*AP*TP*CP*CP*AP*CP*AP*AP*AP*TP*CP*CP*AP*CP*AP*GP*GP*CP*CP*C)-3')


Mass: 7227.697 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#3: DNA chain DNA (41-MER)


Mass: 12855.272 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY

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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: Basal unwinding complex / Type: COMPLEX / Details: Nucleoprotein complex / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 48.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1192718 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320880
ELECTRON MICROSCOPYf_angle_d0.69928512
ELECTRON MICROSCOPYf_dihedral_angle_d17.8953191
ELECTRON MICROSCOPYf_chiral_restr0.0473206
ELECTRON MICROSCOPYf_plane_restr0.0063419

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