6O0Y

Conformational states of Cas9-sgRNA-DNA ternary complex in the presence of magnesium

Summary for 6O0Y

• Entry DOI: 10.2210/pdb6o0y/pdb
• EMDB information: 0584
• Descriptor: CRISPR-associated endonuclease Cas9/Csn1, single guide RNA, 5' product of target strand DNA, ... (5 entities in total)
• Functional Keywords: crispr, cas9, nuclease, genome editing, hydrolase, hydrolase-rna-dna complex, hydrolase/rna/dna
• Biological source: Streptococcus pyogenes; More
• Total number of polymer chains: 5
• Total formula weight: 216628.65

Authors

Zhu, X.,Clarke, R.,Puppala, A.K.,Chittori, S.,Merk, A.,Merrill, B.J.,Simonovic, M.,Subramaniam, S. (deposition date: 2019-02-17, release date: 2019-07-10, Last modification date: 2025-05-28)

Primary citation

Zhu, X.,Clarke, R.,Puppala, A.K.,Chittori, S.,Merk, A.,Merrill, B.J.,Simonovic, M.,Subramaniam, S.
Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.
Nat.Struct.Mol.Biol., 26:679-685, 2019

PubMed Abstract: The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.

PubMed: 31285607
DOI: 10.1038/s41594-019-0258-2

Experimental method

ELECTRON MICROSCOPY (3.37 Å)