Impact of the bridge helix on the conformational flexibility and catalytic activity of Cas12a. (A) and (B) Comparison of the structural rearrangement of the BH and helix 1 relative to the REC domain upon transition from the binary to ternary complex. (A) The structural elements in the binary (PDB: 5NG6) and (B) in the ternary (PDB: 6I1K) FnCas12a complex. The structures were aligned relative to the REC domain as reference point. For clarity, only the bridge helix (BH, green), helix 1 and the REC domain (dark grey) are shown. Tryptophan 971 is highlighted in red and lysine 978 in blue. (C and D) Comparison of the length of BH, helix 1 and connecting linker in the (C) binary (PDB: 5NG6) and (D) ternary complex (PDB: 6I1K). Tryptophan 971 is highlighted in red and lysine 978 in blue. (E) In the apo state, FnCas12a can adopt two conformations: a closed (high FRET population) and an open state (low FRET population). Binding of (pre-)crRNA induces the closure of the enzyme. In order to bind a single-stranded or double-stranded target DNA, Cas12a re-opens again giving rise to a medium FRET population in single-molecule FRET measurements. The bridge helix (BH, in green) is a central structural element in Cas12a that connects the REC and Nuc lobe with the tethering residue tryptophan 971 (shown in red) that anchors the BH in a hydrophobic pocket in the REC lobe. The length of the BH (green), the adjacent helix 1 (gray) and the interconnecting linker (black) as well as the position of W971 re-adjust upon transition from the binary to the ternary complex (see insets). Mutations in the BH or replacement of W971 affect trimming activity of Cas12a and influence the equilibrium between open and closed state of Cas12a.