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- EMDB-5740: Structure of an RNA silencing complex of the CRISPR-Cas immune system -

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Basic information

Entry
Database: EMDB / ID: EMD-5740
TitleStructure of an RNA silencing complex of the CRISPR-Cas immune system
Map data
SampleCmr1-6 effector complex:
Cmr1TRPM8 / Cmr2 / Cmr3 / Cmr4 / Cmr5 / Cmr6
KeywordsEffector complex / RNA cleavage / bacterial immunity
Function / homology
Function and homology information


defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / nucleotide binding / RNA binding / metal ion binding / cytoplasm
CRISPR-associated protein, TM1791 / CRISPR-associated RAMP Cmr4 / Cmr2, N-terminal domain superfamily / CRISPR-associated protein Cmr2, N-terminal / AF1862-like domain superfamily / GGDEF domain / CRISPR type III-associated protein / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr5 / CRISPR-associated protein, TM1793 ...CRISPR-associated protein, TM1791 / CRISPR-associated RAMP Cmr4 / Cmr2, N-terminal domain superfamily / CRISPR-associated protein Cmr2, N-terminal / AF1862-like domain superfamily / GGDEF domain / CRISPR type III-associated protein / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr5 / CRISPR-associated protein, TM1793 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein Cmr2
CRISPR system Cmr subunit Cmr1-1 / CRISPR system Cmr subunit Cmr2 / CRISPR system Cmr subunit Cmr3 / CRISPR system Cmr endoribonuclease Cmr4 / CRISPR system Cmr subunit Cmr5 / CRISPR system Cmr subunit Cmr6
Biological speciesPyrococcus furiosus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 12 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 12, 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: 3.97
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.97
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5740.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.78 Å/pix.
x 120 pix.
= 333.6 Å
2.78 Å/pix.
x 120 pix.
= 333.6 Å
2.78 Å/pix.
x 120 pix.
= 333.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.78 Å
Density
Contour LevelBy AUTHOR: 3.97 / Movie #1: 3.97
Minimum - Maximum-8.774785039999999 - 15.44263649
Average (Standard dev.)0.0 (±0.84591085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-20-20-20
Dimensions120120120
Spacing120120120
CellA=B=C: 333.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z333.600333.600333.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-20-20-20
NC/NR/NS120120120
D min/max/mean-8.77515.443-0.000

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Supplemental data

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Sample components

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Entire Cmr1-6 effector complex

EntireName: Cmr1-6 effector complex / Number of components: 6
MassTheoretical: 350 kDa

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Component #1: protein, Cmr1

ProteinName: Cmr1TRPM8 / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesUniProt: CRISPR system Cmr subunit Cmr1-1

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Component #2: protein, Cmr2

ProteinName: Cmr2 / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesUniProt: CRISPR system Cmr subunit Cmr2

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Component #3: protein, Cmr3

ProteinName: Cmr3 / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesUniProt: CRISPR system Cmr subunit Cmr3

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Component #4: protein, Cmr4

ProteinName: Cmr4 / Number of Copies: 3 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesUniProt: CRISPR system Cmr endoribonuclease Cmr4

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Component #5: protein, Cmr5

ProteinName: Cmr5 / Number of Copies: 3 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesUniProt: CRISPR system Cmr subunit Cmr5

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Component #6: protein, Cmr6

ProteinName: Cmr6 / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)
External referencesUniProt: CRISPR system Cmr subunit Cmr6

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/mL / Buffer solution: 20 mM Tris-HCl, 100 mM NaCl / pH: 7.5
Support filmC-flat 2/2 400 mesh holey carbon grid
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 93 K / Humidity: 100 %
Method: Plasma clean grids for 5 seconds, apply 3 uL sample, and blot for 4 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Nov 2, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 107913 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3000 nm
Specimen HolderHolder: Nitrogen cooled / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 94
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4182

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 388 / Applied symmetry: C1 (asymmetric) / Number of projections: 43749
Details: Images were collected automatically using Leginon software. Particles were automatically picked using a template for single particle reconstruction.
3D reconstructionAlgorithm: Single particle / Software: EMAN, Spider / CTF correction: Phase flip each particle / Resolution: 12 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: flexible / Refinement space: REAL
Details: Cmr2-Cmr3 crystal structure was fit into the EM density using the "fit in map" feature from Chimera. EM density corresponding to Cmr2-Cmr3 was segmented out. The Cmr2-Cmr3 structure was ...Details: Cmr2-Cmr3 crystal structure was fit into the EM density using the "fit in map" feature from Chimera. EM density corresponding to Cmr2-Cmr3 was segmented out. The Cmr2-Cmr3 structure was refined in the Cmr2-Cmr3 density map using MDFF with an EM scaling factor of 0.3 and implicit solvent.
Input PDB model: 4H4K
Chain ID: A, C

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