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Yorodumi- PDB-9ash: Cryo-EM structure of the active Lactococcus lactis Csm bound to t... -
+Open data
-Basic information
Entry | Database: PDB / ID: 9ash | ||||||
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Title | Cryo-EM structure of the active Lactococcus lactis Csm bound to target in post-cleavage stage | ||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA / Type III-A CRISPR-Cas / Csm / Cyclic Oligoadenylate synthesis / RNA BINDING PROTEIN-RNA complex | ||||||
Function / homology | Function and homology information exonuclease activity / endonuclease activity / defense response to virus / RNA binding / ATP binding Similarity search - Function | ||||||
Biological species | Lactococcus lactis subsp. lactis (lactic acid bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.58 Å | ||||||
Authors | Wang, B. / Goswami, H.N. / Li, H. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nucleic Acids Res / Year: 2024 Title: Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production. Authors: Hemant N Goswami / Fozieh Ahmadizadeh / Bing Wang / Doreen Addo-Yobo / Yu Zhao / A Carl Whittington / Huan He / Michael P Terns / Hong Li / Abstract: The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers ...The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers two additional activities: shredding single-stranded DNA and synthesizing cyclic oligoadenylates (cOAs) by the Cas10 subunit. Known Cas10 enzymes exhibit a fascinating diversity in cOA production. Three major forms-cA3, cA4 and cA6have been identified, each with the potential to trigger unique downstream effects. Whereas the mechanism for cOA-dependent activation is well characterized, the molecular basis for synthesizing different cOA isoforms remains unclear. Here, we present structural characterization of a cA6-producing Csm complex during its activation by an activator RNA. Analysis of the captured intermediates of cA6 synthesis suggests a 3'-to-5' nucleotidyl transferring process. Three primary adenine binding sites can be identified along the chain elongation path, including a unique tyrosine-threonine dyad found only in the cA6-producing Cas10. Consistently, disrupting the tyrosine-threonine dyad specifically impaired cA6 production while promoting cA4 production. These findings suggest that Cas10 utilizes a unique enzymatic mechanism for forming the phosphodiester bond and has evolved distinct strategies to regulate the cOA chain length. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 9ash.cif.gz | 555.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb9ash.ent.gz | 446.8 KB | Display | PDB format |
PDBx/mmJSON format | 9ash.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/as/9ash ftp://data.pdbj.org/pub/pdb/validation_reports/as/9ash | HTTPS FTP |
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-Related structure data
Related structure data | 43814 43815 9asiC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-CRISPR system ... , 4 types, 9 molecules AFHGIDECJ
#1: Protein | Mass: 87365.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria) Gene: cas10 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CEJ3 | ||||
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#3: Protein | Mass: 23869.170 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria) Gene: csm3 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CEA3 #5: Protein | Mass: 17014.330 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria) Gene: csm2 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CFW2 #7: Protein | | Mass: 40621.797 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria) Gene: csm5 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CG31 |
-Protein , 1 types, 1 molecules B
#2: Protein | Mass: 33943.277 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria) Gene: csm4 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CFH1 |
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-RNA chain , 3 types, 3 molecules RTP
#4: RNA chain | Mass: 11946.271 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) Lactococcus lactis subsp. lactis (lactic acid bacteria) |
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#6: RNA chain | Mass: 11558.874 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) Lactococcus lactis subsp. lactis (lactic acid bacteria) |
#8: RNA chain | Mass: 1102.620 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria) Production host: Escherichia coli (E. coli) |
-Non-polymers , 3 types, 6 molecules
#9: Chemical | ChemComp-ATP / | ||
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#10: Chemical | #11: Water | ChemComp-HOH / | |
-Details
Has ligand of interest | Y |
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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: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10 Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-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: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
EM software | Name: PHENIX / Version: 1.20.1_4487: / Category: model refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 965374 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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