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Yorodumi- EMDB-6165: Essential Structural and Functional Roles of the Cmr4 Subunit in ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6165 | |||||||||
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Title | Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex | |||||||||
Map data | Reconstruction of Cmr1-6 with crRNA and target RNA | |||||||||
Sample |
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Keywords | CRISPR / Cmr complex / RNA interference / Cmr proteins | |||||||||
Biological species | Pyrococcus furiosus (archaea) / unidentified (others) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 13.0 Å | |||||||||
Authors | Ramia NF / Spilman M / Tang L / Shao Y / Elmore J / Hale C / Cocozaki A / Bhattacharya N / Terns RM / Terns MP ...Ramia NF / Spilman M / Tang L / Shao Y / Elmore J / Hale C / Cocozaki A / Bhattacharya N / Terns RM / Terns MP / Li H / Stagg SM | |||||||||
Citation | Journal: Cell Rep / Year: 2014 Title: Essential structural and functional roles of the Cmr4 subunit in RNA cleavage by the Cmr CRISPR-Cas complex. Authors: Nancy F Ramia / Michael Spilman / Li Tang / Yaming Shao / Joshua Elmore / Caryn Hale / Alexis Cocozaki / Nilakshee Bhattacharya / Rebecca M Terns / Michael P Terns / Hong Li / Scott M Stagg / Abstract: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA ...The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E 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_6165.map.gz | 13.4 MB | EMDB map data format | |
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Header (meta data) | emd-6165-v30.xml emd-6165.xml | 13.5 KB 13.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_6165_fsc.xml | 6.7 KB | Display | FSC data file |
Images | 400_6165.gif 80_6165.gif | 27.9 KB 2.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6165 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6165 | HTTPS FTP |
-Validation report
Summary document | emd_6165_validation.pdf.gz | 78.8 KB | Display | EMDB validaton report |
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Full document | emd_6165_full_validation.pdf.gz | 77.9 KB | Display | |
Data in XML | emd_6165_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6165 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6165 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6165.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Cmr1-6 with crRNA and target RNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 : Cmr complex with crRNA and target RNA
Entire | Name: Cmr complex with crRNA and target RNA |
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Components |
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-Supramolecule #1000: Cmr complex with crRNA and target RNA
Supramolecule | Name: Cmr complex with crRNA and target RNA / type: sample / ID: 1000 Oligomeric state: 1 Cmr1 : 1 Cmr2 : 1 Cmr3 : 4 Cmr4 : 3 Cmr5 : 1 Cmr6 : 1 crRNA : 1 target RNA Number unique components: 8 |
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-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) |
-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) |
-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) |
-Macromolecule #4: Cmr4
Macromolecule | Name: Cmr4 / type: protein_or_peptide / ID: 4 / Number of copies: 4 / Recombinant expression: Yes |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-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) |
-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) |
-Macromolecule #7: crRNA
Macromolecule | Name: crRNA / type: rna / ID: 7 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Pyrococcus furiosus (archaea) |
-Macromolecule #8: target RNA
Macromolecule | Name: target RNA / type: rna / ID: 8 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: unidentified (others) |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 Details: 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5% v/v glycerol, 5 mM beta-mercaptoethanol |
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Staining | Type: NEGATIVE / Details: 2% w/v uranyl formate |
Grid | Details: 400 mesh copper grid with thin carbon support |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification. Legacy - Electron beam tilt params: 0 |
Date | Nov 13, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 4478 / Average electron dose: 15 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |