Summary Bacterial and archaeal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR (cr)RNAs containing the acquired sequences are incorporated into effector complexes that destroy matching invader nucleic acids. The multi-component Cmr effector complex cleaves RNA targets complementary to the crRNAs. Here we report cryo-electron microscopy reconstruction of a functional Cmr complex bound with a target RNA at ∼12Å. Pairs of the Cmr4 and Cmr5 proteins form a helical core that is asymmetrically capped on each end by distinct pairs of the four remaining subunits – Cmr2 and Cmr3 at the conserved 5′ crRNA tag sequence and Cmr1 and Cmr6 near the 3′ end of the crRNA. The shape and organization of the RNA-targeting Cmr complex is strikingly similar to the DNA-targeting Cascade complex. Our results reveal a remarkably conserved architecture among very distantly related CRISPR-Cas complexes.

The effector complex of the Cmr/type III-B CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) system cleaves RNAs recognized by the CRISPR RNA (crRNA) of the complex and includes six protein subunits of unknown functions. Using reconstituted Pyrococcus furiosus Cmr complexes, we found that each of the six Cmr proteins plays a critical role in either crRNA interaction or target RNA capture. Cmr2, Cmr3, Cmr4, and Cmr5 are all required for formation of a crRNA-containing complex detected by native gel electrophoresis, and the conserved 59 repeat sequence tag and 59-OH group of the crRNA are essential for the interaction. Interestingly, capture of the complementary target RNA additionally requires both Cmr1 and Cmr6. In detailed functional studies, we determined that P. furiosus Cmr complexes cleave target RNAs at 6-nucleotide (nt) intervals in the region of complementarity, beginning 5 nt downstream from the crRNA tag and continuing to within~14 nt of the 39 end of the crRNA. Our findings indicate that Cmr3 recognizes the signature crRNA tag sequence (and depends on protein-protein interactions with Cmr2, Cmr4, and Cmr5), each Cmr4 subunit mediates a target RNA cleavage, and Cmr1 and Cmr6 mediate an essential interaction between the 39 region of the crRNA and the target RNA.

Summary Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3Å crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two α-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function, and that the essential role by Cmr2 does not require the identified ADP- or metal-binding, or HD domains in vitro.

Summary The Cmr complex is the multi-subunit effector complex of the Type III-B CRISPR-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and x-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically-packed Cmr4 subunits, which comprise the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr Type III-A and Cascade Type I-E complexes.

Summary The Cmr complex is an RNA-guided effector complex that cleaves invader RNA in the prokaryotic immune response mediated by the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. Here we report the crystal structure of a Cmr subcomplex containing Cmr2 (Cas10) and Cmr3 subunits at 2.8 Å resolution. The structure revealed a dual Ferredoxin fold and glycine-rich loops characteristic of previously known repeat-associated mysterious proteins (RAMPs) and two unique insertion elements in Cmr3 that mediate its interaction with Cmr2. Surprisingly, while mutation of both insertion elements significantly weakened Cmr3-Cmr2 interaction, they exhibit differential effects on Cmr-mediated RNA cleavage by the Cmr complex, suggesting stabilization of Cmr2-Cmr3 interactions by other subunits. Further mutational analysis of the two conserved (but non-Cmr2-binding) glycine-rich loops of Cmr3 identified a region that is likely involved in assembly or the RNA cleavage function of the Cmr complex.