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Yorodumi- EMDB-5315: Structures of the RNA-guided surveillance complex from a bacteria... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5315 | |||||||||
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Title | Structures of the RNA-guided surveillance complex from a bacterial immune system | |||||||||
Map data | reconstruction of the target-bound E. coli Cascade complex | |||||||||
Sample |
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Keywords | Cascade / bacterial immune system / CRISPR / RNA-guided / ribo-nucleoprotein / ssRNA | |||||||||
Biological species | Escherichia coli (E. coli) / unidentified (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 9.2 Å | |||||||||
Authors | Wiedenheft B / Lander GC / Zhou K / Jore MM / Brouns SJJ / van der Oost J / Doudna JA / Nogales E | |||||||||
Citation | Journal: Nature / Year: 2011 Title: Structures of the RNA-guided surveillance complex from a bacterial immune system. Authors: Blake Wiedenheft / Gabriel C Lander / Kaihong Zhou / Matthijs M Jore / Stan J J Brouns / John van der Oost / Jennifer A Doudna / Eva Nogales / Abstract: Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These ...Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5' end of the crRNA. Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences. | |||||||||
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_5315.map.gz | 10.3 MB | EMDB map data format | |
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Header (meta data) | emd-5315-v30.xml emd-5315.xml | 11.2 KB 11.2 KB | Display Display | EMDB header |
Images | emd_5315_1.png | 153.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5315 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5315 | HTTPS FTP |
-Validation report
Summary document | emd_5315_validation.pdf.gz | 78.7 KB | Display | EMDB validaton report |
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Full document | emd_5315_full_validation.pdf.gz | 77.9 KB | Display | |
Data in XML | emd_5315_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5315 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5315 | 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_5315.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | reconstruction of the target-bound E. coli Cascade complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.3 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 : E. coli Cascade complex bound to complementary RNA target
Entire | Name: E. coli Cascade complex bound to complementary RNA target |
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Components |
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-Supramolecule #1000: E. coli Cascade complex bound to complementary RNA target
Supramolecule | Name: E. coli Cascade complex bound to complementary RNA target type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: one asymmetric complex / Number unique components: 2 |
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Molecular weight | Experimental: 415 KDa / Theoretical: 415 KDa / Method: Theoretical |
-Supramolecule #1: Cascade
Supramolecule | Name: Cascade / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Cascade / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Ref GO | 0: GO:0051607 |
Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K12 / synonym: Escherichia coli |
Molecular weight | Experimental: 405 KDa / Theoretical: 405 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: RNA
Macromolecule | Name: RNA / type: rna / ID: 1 / Name.synonym: RNA Details: ssRNA was incubated with the Cascade complex prior to gel filtration Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Experimental: 9.735 KDa / Theoretical: 9.735 KDa |
Sequence | String: TGCCATAACA AGTCTAGGAC CGAACGGTTG TC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.2 mg/mL |
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Buffer | pH: 7.5 / Details: 25 mM HEPES, 100 mM KCl, 1 mM TCEP |
Grid | Details: 200 mesh Cu grid |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 78 K / Instrument: OTHER Details: Vitrification instrument: Vitrobot. blotting at 4 degrees C Method: 4 microliter aliquot of purified sample placed onto C-flat that had been glow-discharged in a nitrogen atmosphere for 60 sec using an Edwards Carbon Evaporator. The grids were blotted for 3 ...Method: 4 microliter aliquot of purified sample placed onto C-flat that had been glow-discharged in a nitrogen atmosphere for 60 sec using an Edwards Carbon Evaporator. The grids were blotted for 3 seconds using a blotting offset of -1. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Min: 78 K / Max: 78 K / Average: 78 K |
Date | Jan 11, 2011 |
Image recording | Category: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 1406 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 100000 |
Sample stage | Specimen holder: Side entry liquid nitrogen-cooled cryo specimen holder Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | From an initial set of 389166 automatically selected particles. Pre-processed with Appion. |
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CTF correction | Details: whole micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 SPARX / Number images used: 176090 |