Database of articles cited by EMDB/PDB/SASBDB data

Keywords
Structure methods	All X-ray diffraction NMR electron microscopy small angle scattering neutron diffraction fiber diffraction powder diffraction infrared spectroscopy EPR fluorescence transfer theoretical model Hybrid
Author
Journal
IF	>30 >20 >10 >5 all

Title	R-loop formation and conformational activation mechanisms of Cas9.
Journal, issue, pages	Nature, Vol. 609, Issue 7925, Page 191-196, Year 2022
Publish date	Aug 24, 2022
Authors	Martin Pacesa / Luuk Loeff / Irma Querques / Lena M Muckenfuss / Marta Sawicka / Martin Jinek /
PubMed Abstract	Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise ...Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.
External links	Nature / PubMed:36002571 / PubMed Central
Methods	EM (single particle) / X-ray diffraction
Resolution	2.54 - 4.14 Å
Structure data	14493 7z4c EMDB-14493, PDB-7z4c: SpCas9 bound to 6 nucleotide complementary DNA substrate Method: EM (single particle) / Resolution: 3.87 Å 14494 7z4e EMDB-14494, PDB-7z4e: SpCas9 bound to 8-nucleotide complementary DNA substrate Method: EM (single particle) / Resolution: 4.14 Å 14496 7z4g EMDB-14496, PDB-7z4g: SpCas9 bound to 12-nucleotide complementary DNA substrate Method: EM (single particle) / Resolution: 3.64 Å 14497 7z4h EMDB-14497, PDB-7z4h: SpCas9 bound to 14-nucleotide complementary DNA substrate Method: EM (single particle) / Resolution: 3.49 Å 14498 7z4i EMDB-14498, PDB-7z4i: SpCas9 bound to 16-nucleotide complementary DNA substrate Method: EM (single particle) / Resolution: 3.12 Å 14499 7z4j EMDB-14499, PDB-7z4j: SpCas9 bound to 18-nucleotide complementary DNA substrate in the catalytic state Method: EM (single particle) / Resolution: 2.99 Å 14500 7z4k EMDB-14500, PDB-7z4k: SpCas9 bound to 10-nucleotide complementary DNA substrate Method: EM (single particle) / Resolution: 3.81 Å 14501 7z4l EMDB-14501, PDB-7z4l: SpCas9 bound to 18-nucleotide complementary DNA substrate in the checkpoint state Method: EM (single particle) / Resolution: 2.54 Å PDB-7z4d: Crystal structure of SpCas9 bound to a 10 nucleotide complementary DNA substrate Method: X-RAY DIFFRACTION / Resolution: 3.1 Å
Chemicals	ChemComp-K: Unknown entry ChemComp-HOH: WATER / Water ChemComp-MG: Unknown entry
Source	streptococcus pyogenes (bacteria) synthetic construct (others)
Keywords	HYDROLASE / CRISPR / Cas9 / R-loop / substrate binding / off-target