[English] 日本語
Yorodumi- EMDB-5929: CasA mediates Cas3-catalyzed target degradation during CRISPR RNA... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5929 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference | |||||||||
Map data | Cryo-EM reconstruction of Cascade bound to a 72 bp target dsDNA | |||||||||
Sample |
| |||||||||
Keywords | Cascade / CRISPR RNA / Cas3 / bacterial immunity | |||||||||
Function / homology | Function and homology information protein-containing complex => GO:0032991 / : / CRISPR-cas system / protein binding / DNA/RNA hybrid binding / maintenance of CRISPR repeat elements / RNA processing / RNA endonuclease activity / endonuclease activity / defense response to virus ...protein-containing complex => GO:0032991 / : / CRISPR-cas system / protein binding / DNA/RNA hybrid binding / maintenance of CRISPR repeat elements / RNA processing / RNA endonuclease activity / endonuclease activity / defense response to virus / nucleic acid binding / Hydrolases; Acting on ester bonds / protein-containing complex / DNA binding / RNA binding / zinc ion binding Similarity search - Function | |||||||||
Biological species | Escherichia coli K-12 (bacteria) / Enterobacteria phage P7 (virus) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 9.0 Å | |||||||||
Authors | Hochstrasser ML / Taylor DW / Bhat P / Guegler CK / Sternberg SH / Nogales E / Doudna JA | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2014 Title: CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference. Authors: Megan L Hochstrasser / David W Taylor / Prashant Bhat / Chantal K Guegler / Samuel H Sternberg / Eva Nogales / Jennifer A Doudna / Abstract: In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA ...In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage. | |||||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5929.map.gz | 175.1 KB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-5929-v30.xml emd-5929.xml | 19.3 KB 19.3 KB | Display Display | EMDB header |
Images | emd_5929.png | 331.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5929 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5929 | HTTPS FTP |
-Validation report
Summary document | emd_5929_validation.pdf.gz | 78.7 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_5929_full_validation.pdf.gz | 77.8 KB | Display | |
Data in XML | emd_5929_validation.xml.gz | 492 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5929 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5929 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_5929.map.gz / Format: CCP4 / Size: 2.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Cryo-EM reconstruction of Cascade bound to a 72 bp target dsDNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.18 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : Cascade bound to a 72 bp target dsDNA
Entire | Name: Cascade bound to a 72 bp target dsDNA |
---|---|
Components |
|
-Supramolecule #1000: Cascade bound to a 72 bp target dsDNA
Supramolecule | Name: Cascade bound to a 72 bp target dsDNA / type: sample / ID: 1000 Oligomeric state: 1 CasA: 2 CasB: 6 CasC: 1 CasD: 1 CasE: 1 crRNA: 1 target dsDNA Number unique components: 7 |
---|---|
Molecular weight | Theoretical: 443.8 KDa |
-Macromolecule #1: CRISPR system Cascade subunit CasA
Macromolecule | Name: CRISPR system Cascade subunit CasA / type: protein_or_peptide / ID: 1 Name.synonym: CRISPR type I-E/Ecoli-associated protein CasA/Cse1, CRISPR-associated protein CasA/Cse1, CasA, Cse1 Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli |
Molecular weight | Theoretical: 55.9 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) |
Sequence | UniProtKB: CRISPR system Cascade subunit CasA GO: defense response to virus, DNA binding, RNA binding, protein-containing complex => GO:0032991 InterPro: CRISPR-associated protein Cse1 |
-Macromolecule #2: CRISPR system Cascade subunit CasB
Macromolecule | Name: CRISPR system Cascade subunit CasB / type: protein_or_peptide / ID: 2 / Name.synonym: CasB, Cse2 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli |
Molecular weight | Theoretical: 18.7 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) |
Sequence | UniProtKB: CRISPR system Cascade subunit CasB GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991 InterPro: CRISPR-associated protein Cse2 |
-Macromolecule #3: CRISPR system Cascade subunit CasC
Macromolecule | Name: CRISPR system Cascade subunit CasC / type: protein_or_peptide / ID: 3 / Name.synonym: CasC, Cas4, Cse4 Details: The 6 CasC subunits make a helical stack forming a groove in which the crRNA lies. Number of copies: 6 / Oligomeric state: helical / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli |
Molecular weight | Theoretical: 40 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) |
Sequence | UniProtKB: CRISPR system Cascade subunit CasC GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991, protein binding InterPro: CRISPR-associated protein, CT1975 |
-Macromolecule #4: CRISPR system Cascade subunit CasD
Macromolecule | Name: CRISPR system Cascade subunit CasD / type: protein_or_peptide / ID: 4 / Name.synonym: CasD, Cas5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli |
Molecular weight | Theoretical: 25.2 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) |
Sequence | UniProtKB: CRISPR system Cascade subunit CasD GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991 InterPro: CRISPR-associated protein, Cas5, CRISPR-associated protein Cas5, N-terminal, CRISPR-associated protein, CasD |
-Macromolecule #5: CRISPR system Cascade subunit CasE
Macromolecule | Name: CRISPR system Cascade subunit CasE / type: protein_or_peptide / ID: 5 Name.synonym: CasE, Cas6e, CasE endoRNase, crRNA endonuclease Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli |
Molecular weight | Theoretical: 22.3 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) |
Sequence | UniProtKB: CRISPR system Cascade subunit CasE GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991, RNA processing, GO: 0090305, endonuclease activity InterPro: CRISPR-associated protein Cse3 |
-Macromolecule #6: R44 CRISPR RNA
Macromolecule | Name: R44 CRISPR RNA / type: rna / ID: 6 / Name.synonym: crRNA / Classification: OTHER / Structure: OTHER / Synthetic?: No |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli |
Molecular weight | Theoretical: 18.6 KDa |
Sequence | String: AUAAACCGAC GGUAUUGUUC AGAUCCUGGC UUGCCAACAG GAGUUCCCCG CGCCAGCGGG G |
-Macromolecule #7: 72 bp dsDNA target with R44 protospacer
Macromolecule | Name: 72 bp dsDNA target with R44 protospacer / type: dna / ID: 7 / Name.synonym: dsDNA target Details: This strand is annealed to the complementary non-target strand to form the dsDNA target. Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
---|---|
Source (natural) | Organism: Enterobacteria phage P7 (virus) |
Molecular weight | Theoretical: 44.4 KDa |
Sequence | String: CATGAGGTCC CTCGTTTAGT CTGTTGGCAA GCCAGGATCT GAACAATACC GTCATCGGAG GTACGATCAA GG |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2.3 mg/mL |
---|---|
Buffer | pH: 7.5 Details: 50 mM HEPES, pH 7.5, 100 mM KCl, 5% glycerol, 0.1 mM EDTA, 1 mM TCEP |
Grid | Details: C-flat 2/2 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 78 K / Instrument: FEI VITROBOT MARK I Method: A 4 uL drop of purified sample was placed onto C-flat grids that had been glow-discharged in a nitrogen atmosphere for 60 seconds using an Edwards Carbon Evaporator. The grids were blotted ...Method: A 4 uL drop of purified sample was placed onto C-flat grids that had been glow-discharged in a nitrogen atmosphere for 60 seconds using an Edwards Carbon Evaporator. The grids were blotted for 4 seconds using a blotting offset of -1 mm. |
-Electron microscopy
Microscope | FEI TECNAI 20 |
---|---|
Temperature | Average: 78 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification. Legacy - Electron beam tilt params: 0 |
Details | Data acquired using Leginon. |
Date | Dec 26, 2012 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3580 / Average electron dose: 20 e/Å2 |
Tilt angle min | 0 |
Tilt angle max | 0 |
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: -2.8 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 100000 |
Sample stage | Specimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | Image pre-processing performed in Appion. Particles were selected using template-based picking in Appion. |
---|---|
CTF correction | Details: Whole micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 280000 |