Figure 1.

System of coupled thermodynamic cycles. Each endpoint is derived from ²⁴ (PDB accession number 3F73). TtAgo is represented as the blue structure, the guide strand in dark gray, and the target stand in light gray. The red-colored base represents a mutation from the sequence present in the crystal structure. Each set of diagonal arrows depicts a binding process. There is a ΔG value associated with each change, depicted by an arrow and numbered for convenience. Introducing a mutation in the endpoints (depicted by the vertical arrows) modulates the free energy change ΔG for that process by some value ΔΔG, which was calculated by thermodynamic integration. By construction, the sum of free energy changes around any closed loop is zero.