|Entry||Database: PDB / ID: 6ny2|
|Title||CasX-gRNA-DNA(45bp) state I|
|Keywords||RNA BINDING PROTEIN/RNA/DNA / CasX / sgRNA / target DNA / CRISPR / RNA BINDING PROTEIN-RNA-DNA complex|
|Function / homology||DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Uncharacterized protein|
Function and homology information
|Biological species||Deltaproteobacteria bacterium (bacteria)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å|
|Authors||Liu, J.J. / Orlova, N. / Nogales, E. / Doudna, J.A.|
|Funding support|| United States, 1items |
|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 / ...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||Molecule: |
Downloads & links
C: DNA target strand
D: DNA Non-target strand
B: RNA (110-MER)
|#1: DNA chain|| |
Mass: 13823.874 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Deltaproteobacteria bacterium (bacteria)
|#2: DNA chain|| |
Mass: 13742.822 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Deltaproteobacteria bacterium (bacteria)
|#3: Protein|| |
Mass: 108092.836 Da / Num. of mol.: 1 / Mutation: D672A, E769A, D935A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deltaproteobacteria bacterium (bacteria)
Gene: DD725_10130 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A357BT59
|#4: RNA chain|| |
Mass: 39272.391 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Deltaproteobacteria bacterium (bacteria)
|Sequence details||The CasX construct has the following sequence: ...The CasX construct has the following sequence: SNAMEKRINK|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: CasX-gRNA-DNA(45bp) state I / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT|
|Source (natural)||Organism: Deltaproteobacteria bacterium (bacteria)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 7.5|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: unspecified|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 46 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149361 / Symmetry type: POINT|
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