Fig. 5 |. The architecture of the DNA-bound TnsC•ATP heptamer and an updated model for transposition mechanism.

a, SEC profiles of TnsC•ATP (red), dsDNA (grey) and TnsC•ATP•dsDNA (green). Inset: SDS–PAGE analysis of the fractions used for cryo-EM. Gel source data are provided in Supplementary Fig. 1. b, Representative 2D class averages from the sample in a reveal apo DNA-free TnsC heptamers (left) and single, double and triple DNA-bound heptamers. c, The final cryo-EM map (left) and ribbon representation (right) of single heptamer TnsC•ATP•DNA. Seven protomers are labelled; DNA is coloured grey, d, Structural alignment of apo and DNA-bound TnsC reveals that the heptamer is distorted after DNA binding, as visualized by tracking the ATP molecules (red or blue) bound to each subunit. Subunit 2 of the DNA-bound heptamer is displaced by 18.5 Å relative to the apo-TnsC ring. For comparison, the helical rise per turn of the bound DNA is around 31 Å. e, Distortion of the DNA-bound heptamer creates a cleft between subunits 1 and 2 (left) that disrupts the ATP binding pocket (bottom right) compared with an undisrupted ATP binding pocket (top right), f, The model for TnsC recruitment and assembly at DNA target sites bound by TniQ–Cascade for type I-F CRISPR-associated transposons (Methods). The PAM is highlighted (yellow), and the hypothesized path of DNA from target-bound TniQ–Cascade to TnsC is indicated by dotted lines. Numbered positions for the target DNA (grey) hybridized to the crRNA (red) are labelled, as are the hypothetical numbered positions from the 3′ edge of the target DNA to the integration site approximately 50 bp downstream. The donor DNA is probably recruited to the bottom (C-terminal) face of the TnsC oligomer through protein–protein interactions with the heteromeric TnsAB transposase. g, Genome-wide accuracy of RNA-guided DNA integration is increased through sequential assembly of the catalytically competent transpososome complex.