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Yorodumi- EMDB-5740: Structure of an RNA silencing complex of the CRISPR-Cas immune system -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5740 | |||||||||
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Title | Structure of an RNA silencing complex of the CRISPR-Cas immune system | |||||||||
Map data | Single particle reconstruction of Cmr effector complex | |||||||||
Sample |
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Keywords | Effector complex / RNA cleavage / bacterial immunity | |||||||||
Function / homology | Function and homology information endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / nucleotide binding / RNA binding / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | Pyrococcus furiosus (archaea) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 12.0 Å | |||||||||
Authors | Spilman MS / Cocozaki AI / Hale C / Shao Y / Ramia NF / Terns R / Terns M / Li H / Stagg SM | |||||||||
Citation | 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5740.map.gz | 5 MB | EMDB map data format | |
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Header (meta data) | emd-5740-v30.xml emd-5740.xml | 14.5 KB 14.5 KB | Display Display | EMDB header |
Images | emd_5740_1.jpg | 72.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5740 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5740 | HTTPS FTP |
-Validation report
Summary document | emd_5740_validation.pdf.gz | 78.6 KB | Display | EMDB validaton report |
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Full document | emd_5740_full_validation.pdf.gz | 77.7 KB | Display | |
Data in XML | emd_5740_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5740 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5740 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5740.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Single particle reconstruction of Cmr effector complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.78 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Cmr1-6 effector complex
Entire | Name: Cmr1-6 effector complex |
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Components |
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-Supramolecule #1000: Cmr1-6 effector complex
Supramolecule | Name: Cmr1-6 effector complex / type: sample / ID: 1000 / Number unique components: 6 |
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Molecular weight | Theoretical: 350 KDa |
-Macromolecule #1: Cmr1
Macromolecule | Name: Cmr1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: CRISPR system Cmr subunit Cmr1-1 |
-Macromolecule #2: Cmr2
Macromolecule | Name: Cmr2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: CRISPR system Cmr subunit Cmr2 |
-Macromolecule #3: Cmr3
Macromolecule | Name: Cmr3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: CRISPR system Cmr subunit Cmr3 |
-Macromolecule #4: Cmr4
Macromolecule | Name: Cmr4 / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Recombinant expression: Yes |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: CRISPR system Cmr endoribonuclease Cmr4 |
-Macromolecule #5: Cmr5
Macromolecule | Name: Cmr5 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Recombinant expression: Yes |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: CRISPR system Cmr subunit Cmr5 |
-Macromolecule #6: Cmr6
Macromolecule | Name: Cmr6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | UniProtKB: CRISPR system Cmr subunit Cmr6 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2 mg/mL |
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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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Average: 94 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification |
Date | Nov 2, 2012 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 4182 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 107913 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Images were collected automatically using Leginon software. Particles were automatically picked using a template for single particle reconstruction. |
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CTF correction | Details: Phase flip each particle |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, Spider / Number images used: 43749 |
Final two d classification | Number classes: 388 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: C |
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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 |