4UN3
Crystal structure of Cas9 bound to PAM-containing DNA target
Summary for 4UN3
Entry DOI | 10.2210/pdb4un3/pdb |
Related | 4UN4 4UN5 |
Descriptor | SGRNA, CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1, TARGET DNA STRAND, ... (6 entities in total) |
Functional Keywords | hydrolase-dna-rna complex, crispr, genome editing, rnp, protein-rna complex, hydrolase/dna/rna |
Biological source | STREPTOCOCCUS PYOGENES More |
Total number of polymer chains | 4 |
Total formula weight | 198180.50 |
Authors | Anders, C.,Niewoehner, O.,Duerst, A.,Jinek, M. (deposition date: 2014-05-25, release date: 2014-07-23, Last modification date: 2024-05-08) |
Primary citation | Anders, C.,Niewoehner, O.,Duerst, A.,Jinek, M. Structural Basis of Pam-Dependent Target DNA Recognition by the Cas9 Endonuclease Nature, 513:569-, 2014 Cited by PubMed Abstract: The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities. PubMed: 25079318DOI: 10.1038/NATURE13579 PDB entries with the same primary citation |
Experimental method | X-RAY DIFFRACTION (2.593 Å) |
Structure validation
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