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Yorodumi- EMDB-3569: 3D reconstruction for the thick CcdA-CcdB-DNA filaments based on ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3569 | |||||||||
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Title | 3D reconstruction for the thick CcdA-CcdB-DNA filaments based on the cryo-electron microscopy dataset. | |||||||||
Map data | 3D reconstruction for the thick CcdA-CcdB-DNA filaments based on the cryo-electron microscopy dataset. | |||||||||
Sample |
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Biological species | Escherichia coli K-12 (bacteria) / Escherichia coli (E. coli) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 25.0 Å | |||||||||
Authors | Vandervelde A / Efremov R / Loris R | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3569.map.gz | 9.8 MB | EMDB map data format | |
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Header (meta data) | emd-3569-v30.xml emd-3569.xml | 16.7 KB 16.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_3569_fsc.xml | 5.4 KB | Display | FSC data file |
Images | emd_3569.png | 33.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3569 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3569 | HTTPS FTP |
-Validation report
Summary document | emd_3569_validation.pdf.gz | 234.3 KB | Display | EMDB validaton report |
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Full document | emd_3569_full_validation.pdf.gz | 233.4 KB | Display | |
Data in XML | emd_3569_validation.xml.gz | 8.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3569 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3569 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3569.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction for the thick CcdA-CcdB-DNA filaments based on the cryo-electron microscopy dataset. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.87 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Thick filaments formed as the complex between DNA, antitoxin CcdA...
Entire | Name: Thick filaments formed as the complex between DNA, antitoxin CcdA and toxin CcdB. |
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Components |
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-Supramolecule #1: Thick filaments formed as the complex between DNA, antitoxin CcdA...
Supramolecule | Name: Thick 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
Supramolecule | Name: antitoxin CcdA and toxin CcdB / type: complex / ID: 2 / Parent: 1 |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Recombinant expression | Organism: Escherichia coli K-12 (bacteria) |
-Supramolecule #3: DNA
Supramolecule | Name: DNA / type: complex / ID: 3 / Parent: 1 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Recombinant expression | Organism: synthetic construct (others) |
-Macromolecule #1: Antitoxin CcdA
Macromolecule | Name: Antitoxin CcdA / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MKQRITVTVD SDSYQLLKAY DVNISGLVST TMQNEARRLR AERWKAENQE GMAEVARFIE MNGSFADENR DW |
-Macromolecule #2: Toxin CcdB
Macromolecule | Name: Toxin CcdB / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MQFKVYTYKR ESRYRLFVDV QSDIIDTPGR RMVIPLASAR LLSDKVSREL YPVVHIGDES WRMMTTDMAS VPVSVIGEEV ADLSHREND IKNAINLMFW GI |
-Macromolecule #3: DNA
Macromolecule | Name: DNA / type: dna / ID: 3 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R2/1 / 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 | ||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: HOMEMADE PLUNGER | ||||||||
Details | The binding reactions occurred at 4degC. DNA: 15 nM CcdA: 10 uM (dimer concentration) CcdB: 10 uM (dimer concentration) DNA was first incubated with CcdA for 15 minutes. After adding CcdB, the resulting mixture was incubated overnight before preparing the EM sample. |
-Electron microscopy
Microscope | JEOL 1400 |
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Image recording | Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.8 µm / Number grids imaged: 1 / Number real images: 77 / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated defocus max: 3.0 µm / Calibrated defocus min: 1.6 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 3.4 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 40000 |
Sample stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |