Figure 4

Mg²⁺-aided conformational transition to catalytic state. (a) Comparison of the representative HNH conformations from the cMD simulations with Mg²⁺ bound (left) and with Mg²⁺ removed (right) at the reaction interface. The bound Mg²⁺ is shown as a cyan sphere and the HNH active residues are represented in a stick model. (b) Scatter plot of the +4P distances to His840 (d _+4P-H840 ) and Asp861 (d _+4P-D861 ) calculated from different sets of cMD simulations. The Cγ and P atoms were selected for measurement. (c) Scatter plot of the distance pair for Ser867/Asn1054 (d _S867-N1054 ) and Ser355/Ser867 (d _S355-S867 ) from different sets of cMD simulations. The Cα atoms were calculated here. The residue pairs of Ser867/Asn1054 and Ser355/Ser867 were used to characterize the conformational states of HNH domain in previous FRET experiments (Supplementary Fig. 3). If available, the corresponding distance pairs obtained from different Cas9 complex crystal structures are mapped on each plot. Of note, in 4UN3, the loop where Asn1054 resides is disordered and we report an average of the distances calculated from respective modeled structures using 4ZT0, 4ZT9 and 5F9R as a template. The pentagrams indicate the catalytic state derived from the conventional ensemble MD (cMD^ens) simulations (an average over 100 data points is reported).