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- EMDB-5737: Structure of an RNA silencing complex of the CRISPR-Cas immune system -

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Basic information

Entry
Database: EMDB / ID: EMD-5737
TitleStructure of an RNA silencing complex of the CRISPR-Cas immune system
Map dataHelical reconstruction of the Cmr4-Cmr5 filament
Sample
  • Sample: Cmr4-Cmr5 filament
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
KeywordsHelical filament
Function / homology
Function and homology information


defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding / cytoplasm
Similarity search - Function
CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR type III-associated protein / RAMP superfamily
Similarity search - Domain/homology
CRISPR system Cmr endoribonuclease Cmr4 / CRISPR system Cmr subunit Cmr5
Similarity search - Component
Biological speciesPyrococcus furiosus (archaea)
Methodhelical reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsSpilman MS / Cocozaki AI / Hale C / Shao Y / Ramia NF / Terns R / Terns M / Li H / Stagg SM
CitationJournal: Mol Cell / Year: 2013
Title: Structure of an RNA silencing complex of the CRISPR-Cas immune system.
Authors: Michael Spilman / Alexis Cocozaki / Caryn Hale / Yaming Shao / Nancy Ramia / Rebeca Terns / Michael Terns / Hong Li / Scott Stagg /
Abstract: Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNAs ...Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNAs (crRNAs) containing the acquired sequences are incorporated into effector complexes that destroy matching invader nucleic acids. The multicomponent Cmr effector complex cleaves RNA targets complementary to the crRNAs. Here, we report cryoelectron microscopy reconstruction of a functional Cmr complex bound with a target RNA at ~12 Å. Pairs of the Cmr4 and Cmr5 proteins form a helical core that is asymmetrically capped on each end by distinct pairs of the four remaining subunits: Cmr2 and Cmr3 at the conserved 5' crRNA tag sequence and Cmr1 and Cmr6 near the 3' end of the crRNA. The shape and organization of the RNA-targeting Cmr complex is strikingly similar to the DNA-targeting Cascade complex. Our results reveal a remarkably conserved architecture among very distantly related CRISPR-Cas complexes.
History
DepositionAug 2, 2013-
Header (metadata) releaseOct 23, 2013-
Map releaseOct 23, 2013-
UpdateNov 6, 2013-
Current statusNov 6, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5737_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5737.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical reconstruction of the Cmr4-Cmr5 filament
Voxel sizeX=Y=Z: 2.78 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-4.98559809 - 6.49716377
Average (Standard dev.)-0.02771433 (±1.04986346)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 266.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z266.880266.880266.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-4.9866.497-0.028

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Supplemental data

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Sample components

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Entire : Cmr4-Cmr5 filament

EntireName: Cmr4-Cmr5 filament
Components
  • Sample: Cmr4-Cmr5 filament
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5

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Supramolecule #1000: Cmr4-Cmr5 filament

SupramoleculeName: Cmr4-Cmr5 filament / type: sample / ID: 1000 / Oligomeric state: helical filament / Number unique components: 2
Molecular weightTheoretical: 520 KDa

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Macromolecule #1: Cmr4

MacromoleculeName: Cmr4 / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr endoribonuclease Cmr4

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Macromolecule #2: Cmr5

MacromoleculeName: Cmr5 / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr subunit Cmr5

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris-HCl, 100 mM NaCl
GridDetails: C-flat 2/2 400 mesh holey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK IV
Method: Plasma clean grids for 5 seconds, apply 3 uL sample, and blot for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureAverage: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DateSep 10, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 268 / Average electron dose: 15 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flip each particle
Final reconstructionApplied symmetry - Helical parameters - Δz: 29.3 Å
Applied symmetry - Helical parameters - Δ&Phi: 48 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, IHRSR
DetailsHelical reconstruction used IHRSR software package combined with EMAN and in-house software

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