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Yorodumi- PDB-6o7e: Cryo-EM structure of Csm-crRNA-target RNA ternary complex in comp... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6o7e | ||||||
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Title | Cryo-EM structure of Csm-crRNA-target RNA ternary complex in complex with AMPPNP in type III-A CRISPR-Cas system | ||||||
Components |
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Keywords | immune system/rna / cryo-EM structure / Csm-crRNA-target RNA ternary complex in complex with AMPPNP / Type III CRISPR-Cas systerm / IMMUNE SYSTEM / immune system-rna complex | ||||||
Function / homology | Function and homology information 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 / identical protein binding Similarity search - Function | ||||||
Biological species | Thermococcus onnurineus (archaea) Thermococcus onnurineus NA1 (archaea) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
Authors | Jia, N. / Patel, D.J. | ||||||
Citation | Journal: Mol Cell / Year: 2019 Title: Second Messenger cA Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path. Authors: Ning Jia / Roger Jones / George Sukenick / Dinshaw J Patel / Abstract: Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair ...Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cA second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppA), and products (cA), to decipher mechanistic aspects of cA formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppA intermediates and subsequent cA formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cA signaling pathway. | ||||||
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 | 6o7e.cif.gz | 406 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6o7e.ent.gz | 320.5 KB | Display | PDB format |
PDBx/mmJSON format | 6o7e.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6o7e_validation.pdf.gz | 951.6 KB | Display | wwPDB validaton report |
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Full document | 6o7e_full_validation.pdf.gz | 970 KB | Display | |
Data in XML | 6o7e_validation.xml.gz | 61.8 KB | Display | |
Data in CIF | 6o7e_validation.cif.gz | 97.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/o7/6o7e ftp://data.pdbj.org/pub/pdb/validation_reports/o7/6o7e | HTTPS FTP |
-Related structure data
Related structure data | 0640MC 0641C 0642C 6o73C 6o74C 6o75C 6o78C 6o79C 6o7bC 6o7dC 6o7hC 6o7iC 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
-Protein , 5 types, 6 molecules ABCDEY
#1: Protein | Mass: 89682.547 Da / Num. of mol.: 1 / Mutation: H14A, D15N Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea) Strain: NA1 / Gene: TON_0893 / Variant: NA1 / Production host: Escherichia coli (E. coli) References: UniProt: B6YWB8, Hydrolases; Acting on ester bonds, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases | ||||
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#2: Protein | Mass: 21210.293 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea) Strain: NA1 / Gene: TON_0894 / Variant: NA1 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWB9 | ||||
#3: Protein | Mass: 32809.012 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea) Strain: NA1 / Gene: TON_0895 / Variant: NA1 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC0 #4: Protein | | Mass: 32345.061 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea) Strain: NA1 / Gene: TON_0896 / Variant: NA1 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC1 #7: Protein | | Mass: 40588.410 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermococcus onnurineus NA1 (archaea) / Strain: NA1 / Gene: TON_0897 / Variant: NA1 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC2*PLUS |
-RNA chain , 2 types, 2 molecules GH
#5: RNA chain | Mass: 12463.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermococcus onnurineus (archaea) |
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#6: RNA chain | Mass: 12750.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermococcus onnurineus (archaea) |
-Non-polymers , 3 types, 8 molecules
#8: Chemical | #9: Chemical | #10: Chemical | |
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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-crRNA-target RNA complex / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT |
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Molecular weight | Value: 0.3 MDa / Experimental value: YES |
Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8.8 / Details: 20 mM Tris-HCl, pH 8.8, 250 mM NaCl, 2 mM DTT |
Buffer component | Formula: Tris |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % |
-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 |
Image recording | Electron dose: 1.35 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
EM software | Name: RELION / Version: 2.1 / Category: 3D reconstruction |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110194 / Symmetry type: POINT |