Journal: Nucleic Acids Research

Article Title: Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes

doi: 10.1093/nar/gkae1277

Figure Lengend Snippet: Cryo-EM structure of the binary EiCsm complex. ( A ) Schematic diagram of the type III-A CRISPR-Cas locus of Ei. ( B ) Cryo-EM map of the binary EiCsm complex (Csm1 1 Csm2 2 Csm3 3 Csm4 1 Csm5 1 stoichiometry). ( C ) Schematic representation of the domain architecture of Csm1 and Csm4 subunits. ( D ) Structure of the Csm1–Csm4 subcomplex binding to the 5′ handle of the crRNA. ( E ) Close-up view of the Palm domain cOA synthetase active site in the EiCsm1 subunit.

Article Snippet: The genes encoding for the components of the Ei (DSM-15952) Csm complex (Csm1, Csm2, Csm3 D32A , Csm4 and Csm5), together with a single spacer-containing CRISPR array, were all cloned into a single p7XNH3 vector (Addgene, #47064) as a polycistronic expression construct using FX cloning ( ).

Techniques: Cryo-EM Sample Prep, CRISPR, Binding Assay

Journal: Nucleic Acids Research

Article Title: Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes

doi: 10.1093/nar/gkae1277

Figure Lengend Snippet: Target RNA binding induces structural changes in the Csm1 Palm domain active site. ( A ) Cryo-EM map of the ternary EiCsm complex (Csm1 1 Csm2 2 Csm3 3 Csm4 1 Csm5 1 stoichiometry) bound to a CTR. ( B ) Close up view of the crRNA:CTR duplex. Flipped-out nucleobases are indicated with asterisks. ( C ) Conformational changes within EiCsm upon CTR binding, depicted as vector representation. ( D ) Structure of the Csm1–Csm4 subcomplex in the CTR-bound state. ( E ) Vector representations of the conformational changes in EiCsm1 upon CTR binding. Cartoon depicts the CTR-bound state of Csm1. ( F ) Structural overlay of the cOA synthetase active site in the binary and CTR-bound ternary EiCsm complexes.

Article Snippet: The genes encoding for the components of the Ei (DSM-15952) Csm complex (Csm1, Csm2, Csm3 D32A , Csm4 and Csm5), together with a single spacer-containing CRISPR array, were all cloned into a single p7XNH3 vector (Addgene, #47064) as a polycistronic expression construct using FX cloning ( ).

Techniques: RNA Binding Assay, Cryo-EM Sample Prep, Binding Assay, Plasmid Preparation