Talos arctica transmission microscope

4 μL of 0.6 mg/mL SEC-purified prespacer-bound or half-integration mimicking substrate-bound Cas4/1-2 complexes were applied to a Quantifoil holey carbon grid (1.2/1.3, 400 mesh) which had been glow-discharged for 30 sec. Grids were blotted for 4 sec at 6 °C, 100% humidity and plunge-frozen in liquid ethane using a Mark IV FEI/Thermo Fisher Vitrobot. Cryo-EM images were collected on a 200 kV Talos Arctica transmission microscope (Thermo Fisher) equipped with a K3 Summit direct electron detector (Gatan). The total exposure time of each movie stack was ~ 3.5 sec, leading to a total accumulated dose of 50 electrons per A ° which fractionated into 50 frames. Dose fractionated super-resolution movie stacks collected from the K3 Summit direct electron detector were 1x binned to a pixel size of 1.234 Å. The defocus value was set between −1.5 μm to −3.5 μm.

4 μL of 0.6 mg/mL SEC-purified complexes were applied to a Quantifoil holey carbon grid (1.2/1.3, 400 mesh), which had been glow-discharged for 30 sec at 30 mA current. Grids were blotted at 8°C for 4 seconds with zero force setting, 100% humidity and plunge-frozen in liquid ethane using a Mark IV FEI/Thermo Fisher Vitrobot. Cryo-EM images were collected on a 200 kV Talos Arctica transmission microscope (Thermo Fisher) equipped with a K3 direct electron detector (Gatan). The total exposure time of each movie stack was ~ 3.5 s, leading to a total accumulated dose of 50 electrons per Å^2 which fractionated into 50 frames. Dose fractionated super-resolution movie stacks collected from the K3 direct electron detector were 2× binned to a pixel size of 1.23 Å. The defocus value was set between −1.0 μm to −2.5 μm.