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

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Basic information

Entry
Database: EMDB / ID: EMD-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
Sample
  • Sample: E. coli Cascade complex
  • Organelle or cellular component: Cascade
KeywordsCascade / bacterial immune system / CRISPR / RNA-guided / ribo-nucleoprotein / ssRNA
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.8 Å
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.
History
DepositionJun 9, 2011-
Header (metadata) releaseJun 13, 2011-
Map releaseSep 12, 2011-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: RCSB / Status: Released

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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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5314_1.png

Downloads & links

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Map

FileDownload / File: emd_5314.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstructed density of the E. coli cascade complex at 8 Angstroms resolution
Voxel sizeX=Y=Z: 2.3 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-13.261092189999999 - 19.555309300000001
Average (Standard dev.)0.03946149 (±0.45303169)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-72-72-72
Dimensions144144144
Spacing144144144
CellA=B=C: 331.19998 Å
α=β=γ: 90.0 °

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
Components
  • Sample: E. coli Cascade complex
  • Organelle or cellular component: Cascade

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Supramolecule #1000: E. coli Cascade complex

SupramoleculeName: E. coli Cascade complex / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: one asymmetric complex / Number unique components: 1
Molecular weightExperimental: 405 KDa / Theoretical: 405 KDa / Method: Theoretical

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Supramolecule #1: Cascade

SupramoleculeName: Cascade / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Cascade / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Ref GOdivclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp ...
divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp kGO3A00516 07ampajax1 classpoptr giGO005160 7ispandiv
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12 / synonym: Escherichia coli
Molecular weightExperimental: 405 KDa / Theoretical: 405 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5 / Details: 25 mM HEPES, 100 mM KCl, 1 mM TCEP
GridDetails: 200 mesh Cu grid
VitrificationCryogen 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.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 100000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 78 K / Max: 78 K / Average: 78 K
DateSep 17, 2010
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 2370 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 SPARX / Number images used: 275573
DetailsFrom an initial set of 498137 automatically selected particles. Pre-processed with Appion.

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