|Entry||Database: EMDB / ID: 5314|
|Title||Structures of the RNA-guided surveillance complex from a bacterial immune system|
|Map data||reconstructed density of the E. coli cascade complex at 8 Angstroms resolution|
|Sample||E. coli Cascade complex:|
|Keywords||Cascade / bacterial immune system / CRISPR / RNA-guided / ribo-nucleoprotein / ssRNA|
|Source||Escherichia coli (E. coli)|
|Method||single particle reconstruction / cryo EM / 8.8 Å resolution|
|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.
|Date||Deposition: Jun 9, 2011 / Header (metadata) release: Jun 13, 2011 / Map release: Sep 12, 2011 / Last update: Jul 17, 2013|
|Structure viewer||EM map: |
Downloads & links
|File||emd_5314.map.gz (map file in CCP4 format, 11665 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.3 Å|
CCP4 map header:
-Entire E. coli Cascade complex
|Entire||Name: E. coli Cascade complex / Details: The sample was monodisperse. / Number of components: 1 / Oligomeric State: one asymmetric complex|
|Mass||Theoretical: 405 kDa / Experimental: 405 kDa / Measured by: Theoretical|
-Component #1: cellular-component, Cascade
|Cellular-component||Name: Cascade / a.k.a: Cascade / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1|
|Mass||Theoretical: 405 kDa / Experimental: 405 kDa|
|Source||Species: Escherichia coli (E. coli) / Strain: K12|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|External references||Gene Ontology: defense response to virus|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 1.2 mg/ml / Buffer solution: 25 mM HEPES, 100 mM KCl, 1 mM TCEP / pH: 7.5|
|Support film||200 mesh Cu grid|
|Vitrification||Instrument: FEI VITROBOT / Cryogen name: ETHANE / Temperature: 78 K / Humidity: 95 %|
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.
Details: Vitrification instrument: Vitrobot. blotting at 4 degrees C
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Sep 17, 2010|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 100000 X (nominal) / Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 800 - 2500 nm|
|Specimen Holder||Holder: Side entry liquid nitrogen-cooled cryo specimen holder|
Model: GATAN LIQUID NITROGEN / Temperature: 78 K ( 78 - 78 K)
|Camera||Detector: GENERIC GATAN (4k x 4k)|
|Image acquisition||Number of digital images: 2370|
|Processing||Method: single particle reconstruction / Number of projections: 275573|
Details: From an initial set of 498137 automatically selected particles. Pre-processed with Appion.
Applied symmetry: C1 (asymmetric)
|3D reconstruction||Algorithm: Projection matching / Software: EMAN2 SPARX / CTF correction: whole micrograph / Resolution: 8.8 Å / Resolution method: FSC 0.5|
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