Table 1.

Cryo-EM Data Collection, 3D Reconstruction, and Model Refinement; Related to Figures 2 and 5

Data Collection	SpyCas9- sgRNA-AcrIIA2	SpyCas9- sgRNA-AcrIIA2b
Grid	CF-2/2–4C-T (Protochips)	CF-2/2–4C-T (Protochips)
EM	Titan Krios 300 kV, K2 Gatan Summit	Titan Krios 300 kV, K2 Gatan Summit
Pixel size (Å)	0.83	1.15
Defocus range (μm)	–0.5 to –2.3	–0.7 to –2.5
Defocus determination	CTFFIND4	CTFFIND4
Particles picked	EMAN and Gautomatch	Gautomatch
Reconstruction (CryoSPARC)
Accuracy of rotations (°)	1.87	2.035
Accuracy of transitions (pixel)	1.150	1.393
Final resolution (Å)	3.4	3.9
Refinement (Phenix)
Map CC (entire box)	0.793	0.798
Map CC (around atoms)	0.776	0.791
RMSD
Bond lengths (Å)	0.01	0.01
Bond angles (°)	1.09	1
Ramachandran Plot
% favored	92.65	91.48
% allowed	7.22	8.52
% outliers	0.13	0.00
Molprobity
Clashscore	5.67	6.05
Accession Numbers
EMDB	9066	9067
PDB	6MCB	6MCC