Cryo arm 300 electron microscope

Colloidal gold particles (15 nm) were mixed with the sample as a fiducial marker before freezing. The frozen grid was examined with a CRYO ARM 300 electron microscope (JEOL, Ltd.) at 300-kV accelerating voltage. Tilt-series images were collected in the range from −60° to +60° with a 2° increment using a low-dose mode, in which the total electron dose for 61 images was less than 100 e⁻/Ų on the specimen. Images were recorded with a K3 camera (Gatan, Inc.) at a nominal magnification of ×15,000 and a pixel size of 3.257 Å on the specimen using the batch tomography procedure of Serial EM software (46 (link)). Image alignment and tomographic reconstruction were performed with IMOD software version 4.7.15 (47 (link)) using fiducial markers. The final tomograms were calculated with the simultaneous iterative reconstruction technique (SIRT) using images of 6.51 Å per pixel after application of a pixel binning of 2. The image segmentation in the 3D reconstructions was performed with Amira version 5.4.5 (Thermo Fisher Scientific).

Cryo-EM data of TIR-APAZ/Ago–gRNA and the SIR2-APAZ/Ago–gRNA–DNA complexes were collected with a Titan Krios microscope (Thermo Fisher Scientific, Waltham, USA) operated at 300 kV and images were collected using EPU^{46 (link)} at a nominal magnification of 105,000× (resulting in a calibrated physical pixel size of 0.85 Å/pixel) with a defocus range of –1.2 μm to –2.2 μm. The images were recorded on a K3 summit electron direct detector in the super-resolution mode at the end of a GIF-Quantum energy filter operated with a slit width of 20 eV. A dose rate of 15 electrons per pixel per second and an exposure time of 2.5 s were used, generating 40 movie frames with a total dose of ~54 e/Ų. A total of 2221 and 3589 movie stacks were collected for TIR-APAZ/Ago–gRNA complex and SIR2-APAZ/Ago–gRNA–DNA complex, respectively.
The grids for TIR-APAZ/Ago–gRNA–DNA were loaded into a CRYO ARM 300 electron microscope (JEOL, Japan) operating at 300 kV with a K3 direct electron detector (Gatan, USA). Cryo-EM images were recorded automatically using Serial-EM software^{47 (link)} in the super-resolution mode with a super-resolution pixel size of 0.475 Å/pixel at defocus values ranging from –0.5 µm to –2.5 µm at a calibrated magnification of 50,000×. Data were collected at a frame rate of 40 frames per second with a total electron dose of 40 e/Ų.

For cryo-EM experiments, 3 μl aliquots of the Gloeobacter PSI trimers (1.68 mg Chl ml⁻¹) were applied to Quantifoil R1.2/1.3, Cu 200 mesh grids pretreated by gold sputtering. Without waiting for incubation, the excess amount of the solution was blotted off for 5 s with a filter paper in an FEI Vitrobot Mark IV at 4°C under 100% humidity. The grids were plunged into liquid ethane cooled by liquid nitrogen and then transferred into a CRYO ARM 300 electron microscope (JEOL) equipped with a cold-field emission gun operated at 300 kV. Zero-energy loss images were recorded at a nominal magnification of ×60,000 on a direct electron detection camera (Gatan K3, AMETEK) with a nominal defocus range of −1.8 to −0.6 μm. One-pixel size corresponded to 0.823 Å. Each image stack was exposed at a dose rate of 17.555 e⁻ Å⁻² s⁻¹ for 4.0 s, and consisted of dose-fractionated 50 movie frames. In total 7,282 image stacks were collected.