|Entry||Database: EMDB / ID: EMD-0454|
|Title||Cryo-EM structure of the T. thermophilus Csm complex bound to target ssRNA|
|Sample||Thermus thermophilus Csm complex bound to target ssRNA:|
|Biological species||Thermus thermophilus (bacteria)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.8 Å|
|Authors||Liu JJ / Liu TY|
|Funding support|| United States, 4 items |
|Citation||Journal: Nat Commun / Year: 2019|
Title: Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.
Authors: Tina Y Liu / Jun-Jie Liu / Abhishek J Aditham / Eva Nogales / Jennifer A Doudna /
Abstract: Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and ...Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.
|Validation Report||PDB-ID: 6o1o|
SummaryFull reportAbout validation report
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_0454.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.08 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Thermus thermophilus Csm complex bound to target ssRNA
|Entire||Name: Thermus thermophilus Csm complex bound to target ssRNA|
Number of components: 2
-Component #1: protein, Thermus thermophilus Csm complex bound to target ssRNA
|Protein||Name: Thermus thermophilus Csm complex bound to target ssRNA|
Recombinant expression: No
|Source||Species: Thermus thermophilus (bacteria)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
-Component #2: nucleic-acid, RNA
|nucleic acid||Name: RNA / Class: RNA / Structure: OTHER / Synthetic: No|
GGAUAUGCGC CUUGACGUGG UCGUCCCCGG GCGCCUUAUC UACGGUAUCA
|Source||Species: Thermus thermophilus (bacteria)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||pH: 7.9|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: DIRECT ELECTRON DE-16 (4k x 4k)|
|Processing||Method: single particle reconstruction / Number of projections: 87321|
|3D reconstruction||Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF|
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