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- EMDB-43815: Cryo-EM structure of the active Lactococcus lactis Csm bound to t... -
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Open data
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Basic information
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Title | Cryo-EM structure of the active Lactococcus lactis Csm bound to target in pre-cleavage stage | |||||||||
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![]() | Type III-A CRISPR-Cas / Csm / Cyclic Oligoadenylate synthesis / RNA BINDING PROTEIN-RNA complex | |||||||||
Function / homology | ![]() exonuclease activity / endonuclease activity / defense response to virus / RNA binding / ATP binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.79 Å | |||||||||
![]() | Wang B / Goswami HN / Li H | |||||||||
Funding support | ![]()
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![]() | ![]() 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
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Downloads & links
-EMDB archive
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Header (meta data) | ![]() | |||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9asiMC ![]() 43814 ![]() 9ashC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
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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
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Sample components
+Entire : Lactococcus lactis Csm CRISPR-Cas complex, ATP bound and Cas10 fl...
+Supramolecule #1: Lactococcus lactis Csm CRISPR-Cas complex, ATP bound and Cas10 fl...
+Macromolecule #1: CRISPR-associated protein Csm4
+Macromolecule #2: CRISPR system Cms endoribonuclease Csm3
+Macromolecule #4: CRISPR system Cms protein Csm2
+Macromolecule #6: CRISPR system Cms protein Csm5
+Macromolecule #7: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1...
+Macromolecule #3: CRISPR RNA
+Macromolecule #5: Target RNA
+Macromolecule #8: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: ADENOSINE MONOPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
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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: ![]() |
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 |
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Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 587955 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |