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- EMDB-5314: Structures of the RNA-guided surveillance complex from a bacteria... -

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Entry
Database: EMDB / ID: 5314
TitleStructures of the RNA-guided surveillance complex from a bacterial immune system
Map datareconstructed density of the E. coli cascade complex at 8 Angstroms resolution
SampleE. coli Cascade complex:
Cascade
KeywordsCascade / bacterial immune system / CRISPR / RNA-guided / ribo-nucleoprotein / ssRNA
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 8.8 Å resolution
AuthorsWiedenheft B / Lander GC / Zhou K / Jore MM / Brouns SJJ / van der Oost J / Doudna JA / Nogales E
CitationJournal: 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.
DateDeposition: Jun 9, 2011 / Header (metadata) release: Jun 13, 2011 / Map release: Sep 12, 2011 / Last update: Jul 17, 2013

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

Fileemd_5314.map.gz (map file in CCP4 format, 11665 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
144 pix
2.3 Å/pix.
= 331.2 Å
144 pix
2.3 Å/pix.
= 331.2 Å
144 pix
2.3 Å/pix.
= 331.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.3 Å
Density
Contour Level:2.0 (by author), 2 (movie #1):
Minimum - Maximum-13.26109219 - 19.55530930
Average (Standard dev.)0.03946149 (0.45303169)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions144144144
Origin-72-72-72
Limit717171
Spacing144144144
CellA=B=C: 331.19998 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.32.32.3
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z331.200331.200331.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-13.26119.5550.039

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Supplemental data

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Sample components

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Entire E. coli Cascade complex

EntireName: E. coli Cascade complex / Details: The sample was monodisperse. / Number of components: 1 / Oligomeric State: one asymmetric complex
MassTheoretical: 405 kDa / Experimental: 405 kDa / Measured by: Theoretical

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Component #1: cellular-component, Cascade

Cellular-componentName: Cascade / a.k.a: Cascade / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 405 kDa / Experimental: 405 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesGene Ontology: defense response to virus

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.2 mg/ml / Buffer solution: 25 mM HEPES, 100 mM KCl, 1 mM TCEP / pH: 7.5
Support film200 mesh Cu grid
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Sep 17, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 100000 X (nominal) / Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 800 - 2500 nm
Specimen HolderHolder: Side entry liquid nitrogen-cooled cryo specimen holder
Model: GATAN LIQUID NITROGEN / Temperature: 78 K ( 78 - 78 K)
CameraDetector: GENERIC GATAN (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2370

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Image processing

ProcessingMethod: 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 reconstructionAlgorithm: Projection matching / Software: EMAN2 SPARX / CTF correction: whole micrograph / Resolution: 8.8 Å / Resolution method: FSC 0.5

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