Extended Data Fig. 10: In-depth analysis of the three snapshots captured from GsuCas4/Cas1-Cas2 programmed with a half-integration mimic.

a. Superposition of cryo-EM reconstructions to reveal the structural differences among three functional states. b. Orientation view of the full integration snapshot for additional interface analysis. The entire leader- repeat DNA is contacted in a quasi-symmetric fashion at the following four regions. c. Contacts from the two Cas1 subunits to the spacer-repeat DNA. The spacer-side DNA density is degenerate and DNA bending is not significant. The leader-recognition α-helix in the catalytic Cas1 is not inserted into the minor groove of the spacer-side DNA. d. The backbone of the central dyad of CRISPR repeat is contacted by the positive charges and a proline-rich motif on the ridge of the Cas2 dimer. e. Immediately adjacent to the catalytic loop, the linker connecting Cas4 to Cas1 is involved in DNA contact. A conserved PRPI motif is exposed upon Cas4 dissociation and is involved in DNA minor groove contact. f. The 4-bp leader region immediately upstream of the CRISPR repeat is favorably recognized and significantly bent upwards by the DNA minor groove insertion of a glycine-rich α-helix in Cas1. As previously revealed, this recognition leads to strong leader-proximal preference at the first half-integration reaction^10,11,12. A pair of inverted repeats is found at the border region of the CRISPR repeat. This inverted repeat is recognized at the major groove region by the catalytic Histidine-containing loop in Cas1¹². g. Overall structure of the “Half-integration, Cas4 still blocking PAM-side” snapshot. This represents an early state, when Cas4 is still engaged in PAM recognition and the spacer-side leader-repeat is not allowed to enter into the integration site. h. The low-resolution EM density defines that the leader-repeat DNA preferentially contact a positively charged patch in Cas1. It should be noted that we are not able to define which specific DNA contact activates Cas4. This will require even higher temporal and spatial resolutions to resolve.