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Yorodumi- EMDB-6122: RNA-targeting by the Type III-A CRISPR-Cas Csm complex of Thermus... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6122 | |||||||||
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Title | RNA-targeting by the Type III-A CRISPR-Cas Csm complex of Thermus thermophilus | |||||||||
Map data | Reconstruction of Csm complex | |||||||||
Sample |
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Keywords | CRISPR RNA-guided targeting complex | |||||||||
Function / homology | Function and homology information phosphoric diester hydrolase activity / exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / ATP binding / metal ion binding Similarity search - Function | |||||||||
Biological species | Thermus thermophilus (bacteria) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 17.0 Å | |||||||||
Authors | Staals RHJ / Zhu Y / Taylor DW / Kornfeld JE / Sharma K / Barendregt A / Koehorst JJ / Vlot M / Neupane N / Varossieau K ...Staals RHJ / Zhu Y / Taylor DW / Kornfeld JE / Sharma K / Barendregt A / Koehorst JJ / Vlot M / Neupane N / Varossieau K / Sakamoto K / Suzuki T / Dohmae N / Yokoyama S / Schaap PJ / Urlaub H / Heck AJR / Nogales E / Doudna JA / Shinkai A / van der Oost J | |||||||||
Citation | Journal: Mol Cell / Year: 2014 Title: RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Authors: Raymond H J Staals / Yifan Zhu / David W Taylor / Jack E Kornfeld / Kundan Sharma / Arjan Barendregt / Jasper J Koehorst / Marnix Vlot / Nirajan Neupane / Koen Varossieau / Keiko Sakamoto / ...Authors: Raymond H J Staals / Yifan Zhu / David W Taylor / Jack E Kornfeld / Kundan Sharma / Arjan Barendregt / Jasper J Koehorst / Marnix Vlot / Nirajan Neupane / Koen Varossieau / Keiko Sakamoto / Takehiro Suzuki / Naoshi Dohmae / Shigeyuki Yokoyama / Peter J Schaap / Henning Urlaub / Albert J R Heck / Eva Nogales / Jennifer A Doudna / Akeo Shinkai / John van der Oost / Abstract: CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type ...CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence. | |||||||||
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_6122.map.gz | 10.7 MB | EMDB map data format | |
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Header (meta data) | emd-6122-v30.xml emd-6122.xml | 15.8 KB 15.8 KB | Display Display | EMDB header |
Images | emd_6122.png | 178.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6122 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6122 | HTTPS FTP |
-Validation report
Summary document | emd_6122_validation.pdf.gz | 79.2 KB | Display | EMDB validaton report |
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Full document | emd_6122_full_validation.pdf.gz | 78.3 KB | Display | |
Data in XML | emd_6122_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6122 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6122 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6122.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Csm complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 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 : Type III-A CRISPR-Cas Csm complex of Thermus thermophilus
Entire | Name: Type III-A CRISPR-Cas Csm complex of Thermus thermophilus |
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Components |
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-Supramolecule #1000: Type III-A CRISPR-Cas Csm complex of Thermus thermophilus
Supramolecule | Name: Type III-A CRISPR-Cas Csm complex of Thermus thermophilus type: sample / ID: 1000 / Details: The sample was monodisperse. Oligomeric state: 1 Csm1: 3 Csm2: 6 Csm3: 2 Csm4: 1 Csm5: 1 crRNA Number unique components: 6 |
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Molecular weight | Experimental: 426 KDa / Method: Native mass spectrometry |
-Macromolecule #1: Csm1
Macromolecule | Name: Csm1 / type: protein_or_peptide / ID: 1 / Name.synonym: Cas10 / Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Thermus thermophilus (bacteria) / Strain: HB8 |
Molecular weight | Theoretical: 90 KDa |
Sequence | UniProtKB: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) GO: metal ion binding, phosphoric diester hydrolase activity InterPro: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 |
-Macromolecule #2: Csm2
Macromolecule | Name: Csm2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Thermus thermophilus (bacteria) / Strain: HB8 |
Molecular weight | Theoretical: 16 KDa |
Sequence | UniProtKB: CRISPR system Cms protein Csm2 / InterPro: CRISPR-associated protein, Csm2 Type III-A |
-Macromolecule #3: Csm3
Macromolecule | Name: Csm3 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Thermus thermophilus (bacteria) / Strain: HB8 |
Molecular weight | Theoretical: 27 KDa |
Sequence | UniProtKB: CRISPR system Cms endoribonuclease Csm3 InterPro: CRISPR-associated RAMP Csm3, CRISPR type III-associated protein |
-Macromolecule #4: Csm4
Macromolecule | Name: Csm4 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Thermus thermophilus (bacteria) / Strain: HB8 |
Molecular weight | Theoretical: 32 KDa |
Sequence | UniProtKB: Uncharacterized protein |
-Macromolecule #5: Csm5
Macromolecule | Name: Csm5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Thermus thermophilus (bacteria) / Strain: HB8 |
Molecular weight | Theoretical: 45 KDa |
Sequence | UniProtKB: CRISPR system Cms protein Csm5 InterPro: CRISPR-associated protein Csm5, CRISPR type III-associated protein |
-Macromolecule #6: CRISPR RNA
Macromolecule | Name: CRISPR RNA / type: rna / ID: 6 / Name.synonym: crRNA / Details: endogenous crRNA of variable lengths / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Thermus thermophilus (bacteria) / Strain: HB8 |
Molecular weight | Theoretical: 14 KDa |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.03 mg/mL |
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Buffer | pH: 7.4 Details: 25 mM HEPES, pH 7.5, 100 mM KCl, 1 mM TCEP, 5% glycerol |
Staining | Type: NEGATIVE Details: Sample was negatively stained with four consecutive droplets of 2% uranyl acetate. |
Grid | Details: 200 mesh Cu grid with thin carbon, glow discharged with a sputter coater |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification. Legacy - Electron beam tilt params: 0 |
Details | Data acquired using Leginon. |
Date | Oct 18, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 420 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 1.4 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
-Image processing
Details | We used a low-pass-filtered E. coli Cascade structure as an initial model for three-dimensional reconstruction using iterative projection matching refinement with libraries from the EMAN2 and SPARX software packages. |
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CTF correction | Details: each micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 60000 |
Final two d classification | Number classes: 200 |