Titan krios cryo electron microscope

The grids were screened using a Talos Glacios cryo-electron microscope (Thermo Fisher Scientific) operated at 200 kV. The grids were imaged in a Titan Krios cryo-electron microscope (Thermo Fisher Scientific) operated at 300 kV at a magnification of 105,000 × (corresponding to a calibrated sampling of 0.82 Å per pixel). Micrographs were recorded by EPU software (Thermo Fisher Scientific) with a Gatan K3 Summit direct electron detector, where each image was composed of 30 individual frames with an exposure time of 3 s and a dose rate of 17.6 electrons per second per Å^{2 (link)}. Finally, a total of 25,306 movie stacks were collected with a defocus range of −0.5–−2.5 μm.

For the resting channel structure at high pH, data were collected on a Titan Krios cryo-electron microscope (ThermoFisher) operated at 300 keV. Images were recorded on a Gatan K3 camera positioned after an energy filter (20 eV slit width) operating in super-resolution mode with a binned pixel size of 0.648 Å. Data were collected with SerialEM (Mastronarde, 2003 (link)) and dose-fractionated to 50 frames for a total exposure time of 2–3 s and a total dose of 40–50 e^- Å⁻².
For the desensitized state structure at pH 7.0, data were recorded on a Titan Krios cryo-electron microscope operated at 300 kV and equipped with a spherical aberration corrector. Images were recorded on a Gatan K2 Summit camera in super-resolution mode with a binned pixel size of 1.096 Å. Data were acquired using Leginon (Suloway et al., 2005 (link)) and dose-fractionated to 48 frames at 0.15 s per frame for a total exposure time of 7.25 s and a total dose of 50 e^- Å⁻².

Three microliters of the SAM-IV riboswitch RNAs at 40 μM were applied onto glow-discharged 200-mesh R2/1 Quantifoil grids. The grids were blotted for 2–4 s and rapidly cryocooled in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at room temperature and ~100% humidity. The samples were screened using a Talos Arctica cryo-electron microscope (Thermo Fisher Scientific) operated at 200 kV. To test the feasibility of cryo-EM for determining RNA structures, the apo SAM-IV riboswitch was first imaged in a Talos Arctica cryo-electron microscope with and without phase plate, respectively. For the high-resolution study, both of the apo and SAM-bound SAM-IV riboswitches were imaged in a Titan Krios cryo-electron microscope (Thermo Fisher Scientific) with GIF energy filter (Gatan) at a magnification of ×130,000 (corresponding to a calibrated sampling of 1.06 Å per pixel). Micrographs were recorded by EPU software (Thermo Fisher Scientific) with a Gatan K2 Summit direct electron detector, where each image was composed of 30 individual frames with an exposure time of 6 s and a dose rate of 7.6 electrons per second per Ų. Finally, a total of 7200 movie stacks for the apo state and 6030 movie stacks for the bound state were collected with a defocus range of −1.5 to −3.5 μm.

Purified protein (3 μL) was applied to glow-discharged Quantifoil R1.2/1.3 grids (Quantifoil, Großlöbichau, Germany) and plunge-frozen in a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA) at 4.5 °C. Images were taken on a Titan Krios cryo-electron microscope (Thermo Fisher Scientific, USA) equipped with a Falcon II direct electron detector (Thermo Fisher Scientific, USA) with an accelerating voltage of 300 kV. A total of 3184 stacks of 20 images each were acquired. The size of the images was 4096 × 4096 pixels at a resolution of 1.107 Å/pix. The electron dose absorbed by each image in the stack was 4 electrons per Ų.