Extended Data Fig. 6: MST and cryo-EM analysis of the binding between CbCas9 and PcrIIC1 in three stages.
From: Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity
a, CbCas9–PcrIIC1 complex. Top: equilibrium dissociation constant (Kd) determined by MST, with 25 nM Red-tris-NTA labelled CbCas9 and varying concentrations of PcrIIC1. Data are presented as mean ± s.d. (n = 3 independent replicates). Graphs are represented as FNorm [‰] against PcrIIC1 concentration. dCbCas9 indicates deactivated CbCas9 (D9A, H837A). Mid: 2D class of particles. Bottom: cryo-EM map after Gaussian filtering (sDev 6) and coloured according to each compartment. Atomic models of proteins were fitted in the semitransparent map. b, CbCas9–PcrIIC1–sgRNA complex. Top: Kd determined by MST. Data are presented as mean ± s.d. (n = 3 independent replicates). Graphs are represented as FNorm [‰] against PcrIIC1 concentration. Mid: 2D class of particles. Bottom: cryo-EM map after Gaussian filtering (sDev 6) and coloured according to each compartment. Atomic models of proteins and nucleic acids were fitted in the semitransparent map. c, CbCas9–PcrIIC1–sgRNA–dsDNA (28-bp) complex. Top: Kd determined by MST. Data are presented as mean ± s.d. (n = 3 independent replicates). Graphs are represented as FNorm [‰] against PcrIIC1 concentration. Mid: 2D class of particles. Bottom: cryo-EM map after Gaussian filtering (sDev 6) and coloured according to each compartment. Atomic models of proteins and nucleic acids were fitted in the semitransparent map.
