|Entry||Database: EMDB / ID: EMD-8988|
|Title||CasX binary complex|
|Sample||CasX binary complex|
|Biological species||Deltaproteobacteria (bacteria)|
|Method||single particle reconstruction / cryo EM / Resolution: 7.5 Å|
|Authors||Liu JJ / Orlova N|
|Citation||Journal: Nature / Year: 2019|
Title: CasX enzymes comprise a distinct family of RNA-guided genome editors.
Authors: Jun-Jie Liu / Natalia Orlova / Benjamin L Oakes / Enbo Ma / Hannah B Spinner / Katherine L M Baney / Jonathan Chuck / Dan Tan / Gavin J Knott / Lucas B Harrington / Basem Al-Shayeb / Alexander Wagner / Julian Brötzmann / Brett T Staahl / Kian L Taylor / John Desmarais / Eva Nogales / Jennifer A Doudna /
Abstract: The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of ...The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_8988.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.15 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire CasX binary complex
|Entire||Name: CasX binary complex / Number of components: 1|
-Component #1: protein, CasX binary complex
|Protein||Name: CasX binary complex / Recombinant expression: No|
|Source||Species: Deltaproteobacteria (bacteria)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||pH: 7.5|
|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: 46 e/Å2 / Illumination mode: SPOT SCAN|
|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: 100000|
|3D reconstruction||Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF|
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