|Entry||Database: EMDB / ID: EMD-0517|
|Title||Big conformation of ssRNA-bound CRISPR_Csm complex|
|Sample||Big conformation of ssRNA-bound CRISPR_Csm complex|
|Biological species||Streptococcus thermophilus (bacteria)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.4 Å|
|Authors||Zhang K / Pintilie G / Li S / Zhu Y / Chiu W / Huang Z|
|Citation||Journal: Cell Res. / Year: 2019|
Title: Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry.
Authors: Minghui Guo / Kaiming Zhang / Yuwei Zhu / Grigore D Pintilie / Xiaoyu Guan / Shanshan Li / Michael F Schmid / Zhuo Ma / Wah Chiu / Zhiwei Huang /
Abstract: The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading ...The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_0517.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.06 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Big conformation of ssRNA-bound CRISPR_Csm complex
|Entire||Name: Big conformation of ssRNA-bound CRISPR_Csm complex / Number of components: 1|
-Component #1: protein, Big conformation of ssRNA-bound CRISPR_Csm complex
|Protein||Name: Big conformation of ssRNA-bound CRISPR_Csm complex / Recombinant expression: No|
|Source||Species: Streptococcus thermophilus (bacteria)|
|Source (engineered)||Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.4 mg/mL / pH: 7.6|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %|
-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: 7 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: single particle reconstruction / Number of projections: 73422|
|3D reconstruction||Software: cryoSPARC / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF|
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