Talos arctica tem

Cryo-EM data was collected on a Talos Arctica TEM (Thermo Fisher) operating at 200 kV in counting mode equipped with a K2 Summit direct electron detector (Gatan, Inc.). Data collection was automated using the Leginon (version 3.3) data collection software^{60 (link)}. Movies were collected at a nominal magnification of 36,000x with a physical pixel size of 1.15 Å pixel⁻¹. A total number of 3448 movies were collected, consisting of 200 ms frames and a total exposure time of ~11.8 s, and resulting in a cumulative exposure of 66 electrons/Ų. Movies were acquired using a nominal defocus range of 0.8–1.2 µm. To improve the Euler distribution of the mt-SerRS-tRNA^Ser(UGA) complex, about one-third of the micrographs were collected at an alpha tilt of 30 degrees. Preprocessing was performed in real-time using Warp (v1.0.7)^{61 (link)} in order to monitor data quality. Particle stacks from Warp were input to cryoSPARC (v2.13)^{62 (link)} for 2D classification (50 classes, 65% inner radius window) to assess variety of collected views.

Cryo-EM data were collected on a Talos Arctica TEM (Thermo Fisher) operating at 200 keV in counting mode equipped with a K2 Summit direct electron detector (Gatan, Inc.). Data collection was automated using the Leginon data collection software^{61 (link)}. Movies were collected at a nominal magnification of ×36,000 with a physical pixel size of 1.15 Å pixel⁻¹. The total number of movies for the tRNA^Ser(GCU)-TL and tRNA^Ser(GCU) datasets were 2498 and 1330, respectively, consisted of 200 ms frames and a total exposure time of ~11.8 s, resulting in a cumulative exposure of 66 electrons/Ų. Movies were acquired using a nominal defocus range of 0.8–1.2 µm. Micrographs for the mSerRS-tRNA^Ser(GCU)-TL complex were collected at 0, 20, and 40 degrees alpha tilt. Preprocessing was performed in real time using Warp^{62 (link)} in order to monitor data quality. Particle stacks from Warp were input to CryoSPARC^{63 (link)} for 2D classification (50 classes, 65% inner radius window) to assess variety of collected views.

Cryo-ET samples were imaged at 300 kV on a Titan Krios TEM (Thermo Fisher Scientific) equipped with a K2-XP direct detector (Gatan, Inc.) and a BioQuantum postcolumn energy filter (Gatan Inc.) with a slit width of 20 eV. Imaging was done at a nominal magnification of 105,000× with a physical pixel size of 1.379 Å/pixel. Using SerialEM (55 (link)), dose-symmetric tilt series (56 (link)) were collected with a tilt angle range between −60° and 60°, 3° increment, and dose fractionation of each tilt image into 10 frames.
SPA samples were imaged at 200 kV on a Talos Arctica TEM (Thermo Fisher Scientific) equipped with a K3 direct detector (Gatan, Inc.) and a BioQuantum energy filter (Gatan Inc.) with a slit width of 20 eV. Imaging was done at a nominal magnification of 63,000× in superresolution mode with a physical pixel size of 1.31 Å/pixel. Using SerialEM (55 (link)), a total of 50 frames were captured as movies. See SI Appendix, Table S1.

Ultrathin sections were mounted on Formvar filmed grids and frozen before the transfer into the transmission electron microscope by the use of a Vitrobot™ Mark IV (Thermo Fisher Scientific, Hillsboro, Oregon; USA). The Vitrobot was operated at 22 °C and at a relative humidity of 100%. The grids were plunged without further treatment into liquid ethane just above its freezing point and transferred in liquid nitrogen. The grids were “clipped” (= inserted in a rigid clip-ring) and transferred under liquid nitrogen into a Talos Arctica™ TEM (Thermo Fisher Scientific) operated at an accelerating voltage of 200 kV and at a nominal magnification of 4300× or 2600×, respectively. Image series in the tilt range of −64°/64° (2° tilt-increments) were recorded at full 4k resolution resulting in a pixel size of 4.91 nm or 8.01 nm, respectively. The defocus value was set to −20 μm. Image stack alignment and 3D reconstruction were performed in the context of the Inspect 3D software V4.3 (Thermo Fisher Scientific). Stacks of 10 image slices from the reconstructed volume were exported for data presentation.