+Open data
-Basic information
Entry | Database: PDB / ID: 6ifk | ||||||
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Title | Cryo-EM structure of type III-A Csm-CTR1 complex, AMPPNP bound | ||||||
Components |
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Keywords | RNA BINDING PROTEIN / Csm complex / Type III-A / CRISPR-Cas system | ||||||
Function / homology | Function and homology information exonuclease activity / transferase activity / defense response to virus / endonuclease activity / RNA binding / ATP binding Similarity search - Function | ||||||
Biological species | Streptococcus thermophilus ND03 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
Authors | You, L. / Ma, J. / Wang, J. / Zhang, X. / Wang, Y. | ||||||
Citation | Journal: Cell / Year: 2019 Title: Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference. Authors: Lilan You / Jun Ma / Jiuyu Wang / Daria Artamonova / Min Wang / Liang Liu / Hua Xiang / Konstantin Severinov / Xinzheng Zhang / Yanli Wang / Abstract: Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, ...Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6ifk.cif.gz | 432.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6ifk.ent.gz | 356.8 KB | Display | PDB format |
PDBx/mmJSON format | 6ifk.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6ifk_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 6ifk_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 6ifk_validation.xml.gz | 63.8 KB | Display | |
Data in CIF | 6ifk_validation.cif.gz | 99.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/if/6ifk ftp://data.pdbj.org/pub/pdb/validation_reports/if/6ifk | HTTPS FTP |
-Related structure data
Related structure data | 9653MC 9654C 9655C 9656C 9657C 9658C 9659C 9660C 6iflC 6ifnC 6ifrC 6ifuC 6ifyC 6ifzC 6ig0C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Type III-A CRISPR-associated protein ... , 2 types, 3 molecules ADC
#1: Protein | Mass: 86976.734 Da / Num. of mol.: 1 / Mutation: D16N Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria) Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M0F3 |
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#2: Protein | Mass: 14848.145 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria) Gene: csm2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M049 |
-Type III-A CRISPR-associated RAMP protein ... , 3 types, 5 molecules GFEBH
#3: Protein | Mass: 24584.855 Da / Num. of mol.: 3 / Mutation: D33N Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria) Gene: csm3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M035 #4: Protein | | Mass: 33828.984 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria) Gene: csm4 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M037 #5: Protein | | Mass: 41102.113 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria) Gene: csm5 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M038 |
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-RNA chain , 2 types, 2 molecules NJ
#6: RNA chain | Mass: 10780.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus ND03 (bacteria) |
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#7: RNA chain | Mass: 13701.244 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus ND03 (bacteria) |
-Non-polymers , 3 types, 5 molecules
#8: Chemical | #9: Chemical | #10: Chemical | ChemComp-ZN / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Csm-CTR1 complex, AMPPNP bound / Type: COMPLEX / Details: type III-A CRISPR-Cas interference complex / Entity ID: #1-#7 / Source: RECOMBINANT |
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Molecular weight | Value: 0.29 MDa / Experimental value: NO |
Source (natural) | Organism: Streptococcus thermophilus ND03 (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 60 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.13_2998: / Classification: refinement |
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CTF correction | Type: NONE |
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61538 / Symmetry type: POINT |