Journal: 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
Deposition
Aug 12, 2013
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Header (metadata) release
Oct 23, 2013
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Map release
Oct 23, 2013
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Update
Nov 6, 2013
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Current status
Nov 6, 2013
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: Cmr1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)
Organism: Pyrococcus furiosus (archaea)
Recombinant expression
Organism: Escherichia coli (E. coli)
Sequence
UniProtKB: CRISPR system Cmr subunit Cmr1-1
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Macromolecule #2: Cmr2
Macromolecule
Name: Cmr2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)
Organism: Pyrococcus furiosus (archaea)
Recombinant expression
Organism: Escherichia coli (E. coli)
Sequence
UniProtKB: CRISPR system Cmr subunit Cmr2
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Macromolecule #3: Cmr3
Macromolecule
Name: Cmr3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)
Organism: Pyrococcus furiosus (archaea)
Recombinant expression
Organism: Escherichia coli (E. coli)
Sequence
UniProtKB: CRISPR system Cmr subunit Cmr3
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Macromolecule #4: Cmr4
Macromolecule
Name: Cmr4 / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Recombinant expression: Yes
Source (natural)
Organism: Pyrococcus furiosus (archaea)
Recombinant expression
Organism: Escherichia coli (E. coli)
Sequence
UniProtKB: CRISPR system Cmr endoribonuclease Cmr4
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Macromolecule #5: Cmr5
Macromolecule
Name: Cmr5 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Recombinant expression: Yes
Source (natural)
Organism: Pyrococcus furiosus (archaea)
Recombinant expression
Organism: Escherichia coli (E. coli)
Sequence
UniProtKB: CRISPR system Cmr subunit Cmr5
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Macromolecule #6: Cmr6
Macromolecule
Name: Cmr6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)
Organism: Pyrococcus furiosus (archaea)
Recombinant expression
Organism: Escherichia coli (E. coli)
Sequence
UniProtKB: CRISPR system Cmr subunit Cmr6
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
2 mg/mL
Buffer
pH: 7.5 / Details: 20 mM Tris-HCl, 100 mM NaCl
Grid
Details: C-flat 2/2 400 mesh holey carbon grid
Vitrification
Cryogen 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
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: C
Software
Name: Chimera
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.
Refinement
Space: REAL / Protocol: FLEXIBLE FIT
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