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- EMDB-3568: 3D reconstruction for the thin CcdA-CcdB-DNA filaments based on t... -

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Basic information

Entry
Database: EMDB / ID: EMD-3568
Title3D reconstruction for the thin CcdA-CcdB-DNA filaments based on the negative stain electron microscopy dataset.
Map data3D reconstruction for the thin CcdA-CcdB-DNA filaments based on the negative stain electron microscopy dataset.
Sample
  • Complex: Thin filaments formed as the complex between DNA, antitoxin CcdA and toxin CcdB.
    • Complex: antitoxin CcdA and toxin CcdB
      • Protein or peptide: Antitoxin CcdA
      • Protein or peptide: Toxin CcdB
    • Complex: DNA
      • DNA: DNA
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (E. coli)
Methodhelical reconstruction / negative staining / Resolution: 23.0 Å
AuthorsVandervelde A / Efremov R / Loris R
CitationJournal: Nucleic Acids Res / Year: 2017
Title: Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon.
Authors: Alexandra Vandervelde / Igor Drobnak / San Hadži / Yann G-J Sterckx / Thomas Welte / Henri De Greve / Daniel Charlier / Rouslan Efremov / Remy Loris / Jurij Lah /
Abstract: Bacteria can become transiently tolerant to several classes of antibiotics. This phenomenon known as persistence is regulated by small genetic elements called toxin-antitoxin modules with intricate ...Bacteria can become transiently tolerant to several classes of antibiotics. This phenomenon known as persistence is regulated by small genetic elements called toxin-antitoxin modules with intricate yet often poorly understood self-regulatory features. Here, we describe the structures of molecular complexes and interactions that drive the transcription regulation of the ccdAB toxin-antitoxin module. Low specificity and affinity of the antitoxin CcdA2 for individual binding sites on the operator are enhanced by the toxin CcdB2, which bridges the CcdA2 dimers. This results in a unique extended repressing complex that spirals around the operator and presents equally spaced DNA binding sites. The multivalency of binding sites induces a digital on-off switch for transcription, regulated by the toxin:antitoxin ratio. The ratio at which this switch occurs is modulated by non-specific interactions with the excess chromosomal DNA. Altogether, we present the molecular mechanisms underlying the ratio-dependent transcriptional regulation of the ccdAB operon.
History
DepositionJan 12, 2017-
Header (metadata) releaseFeb 15, 2017-
Map releaseMar 1, 2017-
UpdateSep 20, 2017-
Current statusSep 20, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3568.png

Downloads & links

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Map

FileDownload / File: emd_3568.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction for the thin CcdA-CcdB-DNA filaments based on the negative stain electron microscopy dataset.
Voxel sizeX=Y=Z: 4.58 Å
Density
Contour LevelBy AUTHOR: 0.0421 / Movie #1: 0.015
Minimum - Maximum-0.02525345 - 0.15503567
Average (Standard dev.)-0.00061731157 (±0.009161664)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-45-45-45
Dimensions909090
Spacing909090
CellA=B=C: 412.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.584.584.58
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z412.200412.200412.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-45-45-45
NC/NR/NS909090
D min/max/mean-0.0250.155-0.001

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Supplemental data

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Sample components

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Entire : Thin filaments formed as the complex between DNA, antitoxin CcdA ...

EntireName: Thin filaments formed as the complex between DNA, antitoxin CcdA and toxin CcdB.
Components
  • Complex: Thin filaments formed as the complex between DNA, antitoxin CcdA and toxin CcdB.
    • Complex: antitoxin CcdA and toxin CcdB
      • Protein or peptide: Antitoxin CcdA
      • Protein or peptide: Toxin CcdB
    • Complex: DNA
      • DNA: DNA

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Supramolecule #1: Thin filaments formed as the complex between DNA, antitoxin CcdA ...

SupramoleculeName: Thin filaments formed as the complex between DNA, antitoxin CcdA and toxin CcdB.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: antitoxin CcdA and toxin CcdB

SupramoleculeName: antitoxin CcdA and toxin CcdB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Antitoxin CcdA

MacromoleculeName: Antitoxin CcdA / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKQRITVTVD SDSYQLLKAY DVNISGLVST TMQNEARRLR AERWKAENQE GMAEVARFIE MNGSFADENR DW

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Macromolecule #2: Toxin CcdB

MacromoleculeName: Toxin CcdB / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQFKVYTYKR ESRYRLFVDV QSDIIDTPGR RMVIPLASAR LLSDKVSREL YPVVHIGDES WRMMTTDMAS VPVSVIGEEV ADLSHREND IKNAINLMFW GI

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Macromolecule #3: DNA

MacromoleculeName: DNA / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString: AATTGT GAT GCTTCTAAAA TTACTAAAAT TGAGGATTTT AATGCTACAA CAATGCC TG CCTCTTCTTA TTTCTCCGGA GATCCGAAAA CCCCAAGTTA CGGATCTT C CTCTCCCTCC GCACAGCGTT ACATCCCGTC AGCACAGCAT GTAGTGCCT CATACAGTTG ...String:
AATTGT GAT GCTTCTAAAA TTACTAAAAT TGAGGATTTT AATGCTACAA CAATGCC TG CCTCTTCTTA TTTCTCCGGA GATCCGAAAA CCCCAAGTTA CGGATCTT C CTCTCCCTCC GCACAGCGTT ACATCCCGTC AGCACAGCAT GTAGTGCCT CATACAGTTG CCCATGGCAC TATATGTTGT GTTGTATCTC TGGACTGTGA TGCGCCGCG CAGGGGCGGA AAACAGCGAT ATGATGATTT TCTCAGCGTT G TACACTTC CGGAAAGTCG TTTATTCAAA TAAAGTCGGA TCCATACGAA AC GGGAATG CGGTAATTAC GCTTTGTTTT TATAAGTCAG ATTTTAATTT TTA TTGGTT AACATAACGA AAGGTAAAAT ACATAAGGCT TACTAAAAGC CAGA TAACA GTATGCGTAT TTGCGCGCTG ATTTTTGCGG TATAAGAATA TATAC TGAT ATGTATACCC GAAGTATGTC AAAAAGAGGT GTGCTATGAA GCAGCG TAT TACAGTGACA GTTGACAGCG ACAGCTATCA GTTGCTCAAG GCATATG AT GTCAATATCT CCGGTCTGGT AAGCACAACC ATGCAGAATG AAGCCCGT C GTCTGCGTGC CGAACGCTGG AAAGCGGAAA ATCAGGAAGG GATGGCTGA GGTCGCCCGG TTTATTGAAA TGAACGGCTC TTTTGCTGAC GAGAACAGGG ACTGGTGAA ATGCAGTTTA AGGTTTACAC CTATAAAAGA GAGAGCCGTT A TCGTCTGT TTGTGGATGT ACAGAGTGAT ATTATTGACA CGCCCGGGCG AC GGATGGT GATCCCCCTG GCCAGTGCAC GTCTGCTGTC AGATAAAGTC TCC CGTGAA CTTTACCCGG TGGTGCATAT CGGGGATGAA AGCTGGCGCA TGAT GACCA CCGATATGGC CAGTGTGCCG GTCTCCGTTA TCGGGGAAGA AGTGG CTGA TCTCAGCCAC CGCGAAAATG ACATCAAAAA CGCCATTAAC C

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormula
30.0 mMTris
200.0 mMNaClSodium chloride
0.5 mMEDTAEthylenediaminetetraacetic acid

Details: Just before staining, the sample was supplemented with 5 mM MgAc2 (final concentration).
StainingType: NEGATIVE / Material: Uranyl formate
Details: The sample was applied to a freshly glow-discharged grid for five minutes after which the grid was washed with 5 mM MgAc2 and adsorbed protein stained with 1% uranyl formate.
GridModel: Electron Microscopy Sciences, continuous carbon/formvar
Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.045 kPa / Details: ELMO glow discharger
DetailsThe binding reactions occurred at 20degC. DNA: 15 nM CcdA: 2.5 uM (dimer concentration) CcdB: 2.5 uM (dimer concentration) DNA was first incubated with CcdA for 15 minutes. After adding CcdB, the resulting mixture was incubated for another 15 minutes before preparing the EM sample.

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Electron microscopy

MicroscopeJEOL 1400
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 1.2 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.4 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number grids imaged: 1 / Number real images: 358 / Average electron dose: 50.0 e/Å2

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Image processing

Segment selectionNumber selected: 8260 / Software - Name: e2helixboxer / Details: Before sorting according to pitch
CTF correctionSoftware - Name: SPARX (ver. find_ctf) / Software - details: home written software
Startup modelType of model: OTHER / Details: Cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPARX
Final reconstructionApplied symmetry - Helical parameters - Δz: 38.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 64.3 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX / Number images used: 2600
FSC plot (resolution estimation)

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