K3 bioquantum direct electron detector

Wild-type SeV virion was propagated in embryonated 9-day-old chicken eggs, as described^{52 (link)}. SeV infected allantoic fluid was harvested and stored under −80 °C until use.
The thawed allantoic fluid was centrifugated for 15 min at 4000×g under 4 °C to remove the crude debris. The supernatant was subjected to another round of centrifugation for 4 h at 48,000×g under 4 °C, and the resultant pellet was resuspended with PBS buffer (50 mM NaH₂PO₄, 50 mM NaCl, pH 7.2) and then loaded onto the 25–65% (w/v) continuous sucrose gradient column. After the 18 h ultracentrifugation at 150,000 g under 4 °C, the visible layer was collected and dialyzed overnight in PBS buffer to remove sucrose. The virion suspension was concentrated and lysed with 2% (v/v) Triton X-100 for 6 h under 4 °C, 3.5 μL of which was immediately applied to cryo-EM sample preparation. 500 good micrographs were collected on Titan Krios G³ⁱ TEM (ThermoFisher Scientific, USA), equipped with a K3 BioQuantum direct electron detector (Gatan, USA),

Suitable grids containing vitrified, UVC-treated V. cholerae were clipped and loaded into a CS-corrected Titan Krios (TFS) equipped with a K2 direct electron detector and a post-column energy filter (Gatan, Inc.) set to zero loss imaging with a 20 eV slit. Targets were chosen based on the presence of the flagellar pole in a hole. This extracellular feature is a good indicator for the presence of the F6 chemotaxis array that is located at the same cell pole. A tilt series of each target was collected using SerialEM set to a dose symmetric tilt scheme between −54° and 54°, with 2° increments, and a pixel size of 3.49 Å^{24 (link),25 (link)}. A defocus of −8 µm and a cumulative dose of 140 e−/Å were used as targets.
For the untreated data set, we used data collected during a previous session. This data was collected on a Titan Krios (TFS) microscope equipped with a K3 BioQuantum direct electron detector and energy filter (Gatan, Inc) set to zero loss imaging with a 20 eV slit. Whole cells in a hole were selected as targets. Tilt series were collected using a bidirectional scheme between −60° and 60° with 2° increments, and a pixel size of 5.86 Å. The target defocus was set to −8 µm and the estimated total dose 170 e−/Å.

An aliquot of 4 μL of PSI–ACPI sample at a Chl concentration of 2.0 mg mL⁻¹ was applied to a freshly glow-discharged holey carbon grid (Quantifoil Au R2/1, 200 mesh) with continuous carbon support. The grid was blotted for 2 s at 100% humidity at 8 °C with a force level of 2 and immediately plunged into liquid ethane cooled by liquid nitrogen with Vitrobot Mark IV (Thermo Fisher, USA). The grids were loaded into a 300 kV Titan Krios G³ⁱ microscope (Thermo Fisher) equipped with a K3 BioQuantum direct electron detector (Gatan, USA) for data acquisition. A total of 9,688 movie stacks were automatically recorded using EPU (Thermo Fisher) (Thompson et al. 2019 (link)) at a total dose for a stack of 50 e⁻ Å⁻² in a defocus range of −1.0 to −1.8 μm. A super-resolution mode was used at a nominal magnification of ×81,000 corresponding to a pixel size of 0.53 Å with the energy filter slit set to 20 eV.

Tomograms were collected for an RNA polymerase (0.3 mg/ml) grid using a Thermo Fisher Titan Krios TEM operating at 300 keV at the Pacific Northwest Center for Cryo-EM (PNCC). Gatan K3 BioQuantum direct electron detector equipped with an energy filter operated in zero-loss mode (with slit width of 20 eV) was used. The tomography data were acquired from specific regions of the grid that were not previously exposed and were similar in vitrification quality to the squares used for single-particle data collection. The software SerialEM^{73 (link)} was utilized for data collection, employing a dose-symmetric scheme starting at 0° and capturing alternating negative and positive tilts in 3° increments. The tilt angles for the tomograms ranged from −48° to +54°, while the nominal defocus range during tilt series collection was −2 to −4 µm. Each tilt was recorded as movie frames with 10 sub-frames in counting mode, utilizing a per-frame dose of approximately 0.23 e/Ų. The magnification was set to 42Kx, resulting in a pixel size of 1.06 Å/pixel. A total of 25 tomograms were collected for the RNA polymerase sample, and among them, 5 with best alignment statistics were subjected to further processing and data analysis.