EMDB/PDB/SASBDBから引用されている文献のデータベース

キーワード
構造解析手法	All X線回折 NMR 電子顕微鏡小角散乱中性子線回折線維回折粉末回折赤外分光 EPR FRET 理論モデルハイブリッド
著者・登録者
ジャーナル
IF	>30 >20 >10 >5 all

タイトル	R-loop formation and conformational activation mechanisms of Cas9.
ジャーナル・号・ページ	Nature, Vol. 609, Issue 7925, Page 191-196, Year 2022
掲載日	2022年8月24日
著者	Martin Pacesa / Luuk Loeff / Irma Querques / Lena M Muckenfuss / Marta Sawicka / Martin Jinek /
PubMed 要旨	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.
リンク	Nature / PubMed:36002571 / PubMed Central
手法	EM (単粒子) / X線回折
解像度	2.54 - 4.14 Å
構造データ	14493 7z4c EMDB-14493, PDB-7z4c: SpCas9 bound to 6 nucleotide complementary DNA substrate 手法: EM (単粒子) / 解像度: 3.87 Å 14494 7z4e EMDB-14494, PDB-7z4e: SpCas9 bound to 8-nucleotide complementary DNA substrate 手法: EM (単粒子) / 解像度: 4.14 Å 14496 7z4g EMDB-14496, PDB-7z4g: SpCas9 bound to 12-nucleotide complementary DNA substrate 手法: EM (単粒子) / 解像度: 3.64 Å 14497 7z4h EMDB-14497, PDB-7z4h: SpCas9 bound to 14-nucleotide complementary DNA substrate 手法: EM (単粒子) / 解像度: 3.49 Å 14498 7z4i EMDB-14498, PDB-7z4i: SpCas9 bound to 16-nucleotide complementary DNA substrate 手法: EM (単粒子) / 解像度: 3.12 Å 14499 7z4j EMDB-14499, PDB-7z4j: SpCas9 bound to 18-nucleotide complementary DNA substrate in the catalytic state 手法: EM (単粒子) / 解像度: 2.99 Å 14500 7z4k EMDB-14500, PDB-7z4k: SpCas9 bound to 10-nucleotide complementary DNA substrate 手法: EM (単粒子) / 解像度: 3.81 Å 14501 7z4l EMDB-14501, PDB-7z4l: SpCas9 bound to 18-nucleotide complementary DNA substrate in the checkpoint state 手法: EM (単粒子) / 解像度: 2.54 Å PDB-7z4d: Crystal structure of SpCas9 bound to a 10 nucleotide complementary DNA substrate 手法: X-RAY DIFFRACTION / 解像度: 3.1 Å
化合物	ChemComp-K: Unknown entry / カリウムカチオン ChemComp-HOH: WATER ChemComp-MG: Unknown entry / マグネシウムジカチオン
由来	streptococcus pyogenes (化膿レンサ球菌) synthetic construct (人工物)
キーワード	HYDROLASE / CRISPR / Cas9 / R-loop / substrate binding / off-target