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Yorodumi- EMDB-43814: Cryo-EM structure of the active Lactococcus lactis Csm bound to t... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-43814 | |||||||||
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Title | Cryo-EM structure of the active Lactococcus lactis Csm bound to target in post-cleavage stage | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Type III-A CRISPR-Cas / Csm / Cyclic Oligoadenylate synthesis / RNA BINDING PROTEIN-RNA complex | |||||||||
Function / homology | Function and homology information exonuclease activity / defense response to virus / endonuclease activity / RNA binding / ATP binding Similarity search - Function | |||||||||
Biological species | Lactococcus lactis subsp. lactis (lactic acid bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.58 Å | |||||||||
Authors | Wang B / Goswami HN / Li H | |||||||||
Funding support | United States, 1 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_43814.map.gz | 157.1 MB | EMDB map data format | |
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Header (meta data) | emd-43814-v30.xml emd-43814.xml | 22.5 KB 22.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_43814_fsc.xml | 11.5 KB | Display | FSC data file |
Images | emd_43814.png | 96.2 KB | ||
Filedesc metadata | emd-43814.cif.gz | 7.2 KB | ||
Others | emd_43814_half_map_1.map.gz emd_43814_half_map_2.map.gz | 154.5 MB 154.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-43814 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-43814 | HTTPS FTP |
-Validation report
Summary document | emd_43814_validation.pdf.gz | 846.6 KB | Display | EMDB validaton report |
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Full document | emd_43814_full_validation.pdf.gz | 846.2 KB | Display | |
Data in XML | emd_43814_validation.xml.gz | 20.5 KB | Display | |
Data in CIF | emd_43814_validation.cif.gz | 26.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43814 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43814 | HTTPS FTP |
-Related structure data
Related structure data | 9ashMC 9asiC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_43814.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.074 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_43814_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_43814_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10
+Supramolecule #1: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10
+Macromolecule #1: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1...
+Macromolecule #2: CRISPR-associated protein Csm4
+Macromolecule #3: CRISPR system Cms endoribonuclease Csm3
+Macromolecule #5: CRISPR system Cms protein Csm2
+Macromolecule #7: CRISPR system Cms protein Csm5
+Macromolecule #4: CRISPR RNA
+Macromolecule #6: Target RNA
+Macromolecule #8: RNA (5'-D(*(ATP))-R(P*AP*A)-3')
+Macromolecule #9: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #10: MAGNESIUM ION
+Macromolecule #11: water
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |