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4UN3

Crystal structure of Cas9 bound to PAM-containing DNA target

Summary for 4UN3
Entry DOI10.2210/pdb4un3/pdb
Related4UN4 4UN5
DescriptorSGRNA, CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1, TARGET DNA STRAND, ... (6 entities in total)
Functional Keywordshydrolase-dna-rna complex, crispr, genome editing, rnp, protein-rna complex, hydrolase/dna/rna
Biological sourceSTREPTOCOCCUS PYOGENES
More
Total number of polymer chains4
Total formula weight198180.50
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 citationAnders, 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: 25079318
DOI: 10.1038/NATURE13579
PDB entries with the same primary citation
Experimental method
X-RAY DIFFRACTION (2.593 Å)
Structure validation

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