Fig. 2: Kinetics-guided cryo-EM data analysis.
From: Visualization of a multi-turnover Cas9 after product release
a Data collection conditions used for cryo-EM. The pre-formed Cas9 RNP containing a 17-bp R-loop with 2 terminal mismatches was incubated with a 55-bp dsDNA target for 2 h at 37 °C before vitrification. Representative 2D classes are shown from the final particle stack used for further 3D classification. 3D class distribution represented as a pie chart. b Ensemble of cryo-EM reconstructions generated using 3D classification. c Representative maps corresponding to five main conformational states that span the entire reaction cycle from target binding to product dissociation. d Structures of each state corresponding to the maps shown in panel c. Colored according to the schematic below. e Cas9 architecture colored by domain.
