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- EMDB-16229: Cryo-EM structure of the bacterial replication origin opening bas... -

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Basic information

Entry
Database: EMDB / ID: EMD-16229
TitleCryo-EM structure of the bacterial replication origin opening basal unwinding system
Map data
Sample
  • Complex: Basal unwinding complex
    • Protein or peptide: Chromosomal replication initiator protein DnaA
    • DNA: 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')
    • DNA: DNA (41-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsDnaA / cryo-EM / BUS complex / replication initiation / REPLICATION
Function / homology
Function and homology information


regulation of DNA replication / DNA replication origin binding / DNA replication initiation / ATP hydrolysis activity / ATP binding / plasma membrane / 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 ATPAse domain / 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
Chromosomal replication initiator protein DnaA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria) / DNA molecule (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPelliciari S / Bodet-Lefevre S / Murray H / Ilangovan A
Funding support United Kingdom, 1 items
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, 2022-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16229.png

Downloads & links

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Map

FileDownload / File: emd_16229.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 320 pix.
= 343.04 Å		1.07 Å/pix.
x 320 pix.
= 343.04 Å		1.07 Å/pix.
x 320 pix.
= 343.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.072 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.36148095 - 1.4240421
Average (Standard dev.)0.0008227174 (±0.0343295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 343.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16229_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16229_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Basal unwinding complex

EntireName: Basal unwinding complex
Components
  • Complex: Basal unwinding complex
    • Protein or peptide: Chromosomal replication initiator protein DnaA
    • DNA: 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')
    • DNA: DNA (41-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Basal unwinding complex

SupramoleculeName: Basal unwinding complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: Nucleoprotein complex
Source (natural)Organism: Bacillus subtilis (bacteria)

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Macromolecule #1: Chromosomal replication initiator protein DnaA

MacromoleculeName: Chromosomal replication initiator protein DnaA / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 50.92998 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MENILDLWNQ ALAQIEKKLS KPSFETWMKS TKAHSLQGDT LTITAPNEFA RDWLESRYLH LIADTIYELT GEELSIKFVI PQNQDVEDF MPKPQVKKAV KEDTSDFPQN MLNPKYTFDT FVIGSGNRFA HAASLAVAEA PAKAYNPLFI YGGVGLGKTH L MHAIGHYV ...String:
MENILDLWNQ ALAQIEKKLS KPSFETWMKS TKAHSLQGDT LTITAPNEFA RDWLESRYLH LIADTIYELT GEELSIKFVI PQNQDVEDF MPKPQVKKAV KEDTSDFPQN MLNPKYTFDT FVIGSGNRFA HAASLAVAEA PAKAYNPLFI YGGVGLGKTH L MHAIGHYV IDHNPSAKVV YLSSEKFTNE FINSIRDNKA VDFRNRYRNV DVLLIDDIQF LAGKEQTQEE FFHTFNTLHE ES KQIVISS DRPPKEIPTL EDRLRSRFEW GLITDITPPD LETRIAILRK KAKAEGLDIP NEVMLYIANQ IDSNIRELEG ALI RVVAYS SLINKDINAD LAAEALKDII PSSKPKVITI KEIQRVVGQQ FNIKLEDFKA KKRTKSVAFP RQIAMYLSRE MTDS SLPKI GEEFGGRDHT TVIHAHEKIS KLLADDEQLQ QHVKEIKEQL K

UniProtKB: Chromosomal replication initiator protein DnaA

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Macromolecule #2: DNA (5'-D(P*AP*CP*TP*TP*AP*TP*CP*CP*AP*CP*AP*AP*AP*TP*CP*CP*AP*CP...

MacromoleculeName: 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')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 7.227697 KDa
SequenceString:
(DA)(DC)(DT)(DT)(DA)(DT)(DC)(DC)(DA)(DC) (DA)(DA)(DA)(DT)(DC)(DC)(DA)(DC)(DA)(DG) (DG)(DC)(DC)(DC)

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Macromolecule #3: DNA (41-MER)

MacromoleculeName: DNA (41-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 12.855272 KDa
SequenceString:
(DT)(DA)(DG)(DT)(DA)(DG)(DA)(DA)(DG)(DT) (DA)(DA)(DT)(DA)(DG)(DT)(DA)(DG)(DG)(DG) (DC)(DC)(DT)(DG)(DT)(DG)(DG)(DA)(DT) (DT)(DT)(DG)(DT)(DG)(DG)(DA)(DT)(DA)(DA) (DG) (DT)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 7 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 48.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Abinitio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1192718
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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