6WC0

Crystal structure of AceCas9 bound with guide RNA and DNA with 5'-NNNTC-3' PAM

Summary for 6WC0

• Entry DOI: 10.2210/pdb6wc0/pdb
• Related: 6WBR
• Descriptor: CRISPR-associated endonuclease, Csn1 family, sgRNA (95-MER), DNA (30-MER), ... (4 entities in total)
• Functional Keywords: dna endonuclease, crispr-cas9, hnh, ruvc, rna binding protein, rna binding protein-rna-dna complex, rna binding protein/rna/dna
• Biological source: Acidothermus cellulolyticus (strain ATCC 43068 / 11B); More
• Total number of polymer chains: 4
• Total formula weight: 152255.02

Authors

Li, H.,Das, A. (deposition date: 2020-03-28, release date: 2020-11-18, Last modification date: 2023-10-18)

Primary citation

Das, A.,Hand, T.H.,Smith, C.L.,Wickline, E.,Zawrotny, M.,Li, H.
The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.
Nat Commun, 11:6346-6346, 2020

PubMed Abstract: Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.

PubMed: 33311465
DOI: 10.1038/s41467-020-20204-1

Experimental method

X-RAY DIFFRACTION (3.61 Å)