CRISPR type I-E (Cascade)
SEE CRISPR-Cas Contents
Cascade/I-E from Escherichia coliCrystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target[1]The crystal structure of ssDNA-bound Cascade has the seahorse architecture (4qyz). The body is formed by a helical filament of six Cas7 subunits (Cas7.1 to 7.6) wrapped around the crRNA guide, with a head-to-tail dimer of Cse2 (Cse2.1 and Cse2.2) at the belly. Cas6e and the 3′ handle of crRNA cap the Cas7 filament at the head while Cas5 and the 5′ handle cap the tail. The N-terminal base of Cse1 is positioned at the tail of the filament; the C-terminal four-helix bundle contacts Cse2.2. The ssDNA target is juxtaposed to the guide region of the crRNA in a groove formed by the Cas7 filament, the four-helix bundle of Cse1, and the Cse2 dimer. The two strands of the guide-target hybrid do not twist around one another in a helix, but instead adopt an underwound ribbon-like structure reminiscent of a ladder. The 5′ and 3′ ends of the curved target strand are ~102Å apart, roughly the length of straight B-form dsDNA with an identical sequence (~107 Å). The crRNA (green) and ssDNA target (orange) are displayed in a spheres representation. Underwinding is facilitated by kinks that occur every sixth base pair in the backbone of both strands of the hybrid (ribbon representation of the crRNA and ssDNA). At each kink, complementary nucleotides are rotated ~90°, in opposing directions, from the axis of the duplex. Disrupted RNA and DNA nucleotides are colored red and blue, respectively. Structure of the Cas7 subunit.Within Cascade, the six Cas7 subunits form a right-handed filament, with a pitch of ~135Å, around the guide target hybrid. Rocket representation of one Cas7 colored by domain: thumb (green), fingers (blue), and palm (purple). The filament is arranged such that the thumb of one Cas7 subunit, composed of an extended β hairpin, extends toward the fingers of the adjacent subunit. 90° rotation about axis Z. Stabilization of the guide-target hybrid by Cas7, Cse1, and Cse2.Each 5-bp segment of the hybrid is situated between the palm of one Cas7 subunit (e.g. Cas7.2) and the fingers of the adjacent subunit (e.g. Cas7.3). 5-bp segment is colored red. Extensive contacts between the guide region of the crRNA and the Cas7 filament bury a large portion of the crRNA backbone, leaving the bases solvent-exposed. The absence of direct contacts between protein side chains and bases of the crRNA explains the lack of sequence specificity by Cascade for the guide sequence. Close-up view of the bound crRNA. The DNA target has been removed for clarity. Intercalation by Met166 from Cas7 is highlighted. Several highly conserved polar and positively charged residues (Arg20, Lys27, Ser40, Gln42, Lys45, and Lys49 - colored in magenta) from the palm of one Cas7 (e.g. Cas7.3) contact the RNA backbone, while the fingers from the adjacent Cas7 (e.g. Cas7.2) subunit (residues 109-111, 163-169, colored in plum) contact both strands of the hybrid across the minor groove. Of note, the Thumb (colored in olive) of one Cas7 subunit (e.g. Cas7.4) pushes through the guide-target hybrid at the 1-bp gaps. Representation of 5 thumbs protruding guide-target hybrid at the 1-bp gaps. Each displaced RNA nucleotide adopts the syn conformation, is similarly positioned above the backbone of the downstream RNA, and is contacted by residues from both the Cas7 palm (e.g. Cas7.3 palm; Ser43 and Arg46) and thumb (e.g. Cas7.4 thumb; Thr201 and Val203). Overview of the interactions between the ssDNA target and Cse2.1, Cse2.2, and Cse1. The proteins are represented as rockets, the DNA as a surface. The positions of the disrupted DNA nucleotides (royal blue) are indicated. Interactions capping the tail of CascadeCas5 caps the tail of the Cas7 filament at the 5′ end of the crRNA. The structure of Cascade reveals that Cas5 is structurally related to Cas7, as it consists of a palm(residues 1 to 78 and 115 to 224) and a thumb (residues 79 to 114) domain, but lacks a fingers domain. Rocket representation of Cas7 and Cas5 colored by domain: thumb (green), fingers (blue), and palm (purple). Close-up view of the interaction between Cas5, Cas7.6, and the 5′ hook. Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution[2]Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target (5h9f).
Cascade proteins from E. coli: CasA (Cse1), CasB (Cse2) (2u), CasC (Cas4, Cas7, Cse4) (6u), CasD (Cas5), CasE (Cas6e). Other representatives from E. coli: 4tvx, 4u7u, 5h9e, 5h9f, 5cd4 EcoCas6e (CasE) from Escherichia coli. 4dzd (monomer)I-E Cascade from Thermobifida fuscaType I-E Cascade/R-loop/Cas3 complex from Thermobifida fusca
Cascade proteins from Thermobifida fusca: CRISPR-associated helicase Cas3, Cas5e, Cse1, Cse3, Cse4, uncharacterized protein. Other representatives from T. fusca
Additional representatives of CRISPR type I-ETthCas6e (TTHB192, Cse3) from Thermus thermophilus. CRISPR endoribonuclease TthCas6e (Cse3) bound to 20 nt RNA (2y8w). Other representatives of CRISPR type I-E structures: 1wj9, 2y8y, 2y9h, 3qrp, 3qrq, 3qrr, 4h7a, 4h79.
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