Titan krios microscope

To prepare grids used in the collection of datasets at a 25° tilt, 3.5 µl (0.1 mg/ml) of ISG65:C3b complex was applied to glow-discharged copper C/Flat, 300 mesh 1.2/1.3, and 2/2 TEM grids (Protochips). The grids were vitrified by being plunged into liquid ethane using the Thermo Scientific Vitrobot Mark IV system (4 °C, 100% relative humidity, 30 s waiting time, 4 s blotting time) and then transferred to a Titan Krios microscope (Thermo Fisher Scientific) for data acquisition.
For data collections at a 0° stage tilt, Au, 300 mesh, R1.2/1.3 TEM grids (Protochips) were coated with a graphene monolayer using an in-house developed protocol. About 3.5 µl (0.15 mg/ml) of ISG65:C3b complex was applied to freshly plasma-cleaned TEM grids and vitrified as explained for grids used to obtain the 25° tilt dataset. The grids were subsequently transferred to a Titan Krios microscope (Thermo Fisher Scientific) for data acquisition.
The data were collected at 300 kV using SerialEM^{56 (link)} software. The data were collected on a K2 direct electron detection camera positioned behind a Gatan Imaging Filter (Bioquantum 967, Gatan). The camera was operated in the electron counting mode, and the data were collected at the calibrated pixel size of 0.818 Å/px. The data from a 5.0 s exposure were split into 40 frames comprising an overall dose of 60 e⁻/Ų.

3.5 μL of afTMEM16-containing nanodiscs (7 mg mL⁻¹) supplemented with 3 mM Fos-Choline-8-Fluorinated (Anatrace) was applied to a glow-discharged UltrAuFoil R1.2/1.3 300-mesh gold grid (Quantifoil) and incubated for one minute under 100% humidity at 15°C. Following incubation, grids were blotted for 2 s and plunge frozen in liquid ethane using a Vitrobot Mark IV (FEI). For the +C24:0 Ceramide/+Ca²⁺and EDTA samples as well as +Ca²⁺Dataset D (Supplementary file 1) micrographs were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a K2 Summit direct electron detector (Gatan), using a slid width of 20 eV on a GIF Quantum energy filter and a Cs corrector with a calibrated pixel size of 1.0961 Å/pixel. A total dose of 62.61 e^–/Ų distributed over 45 frames (1.39 e^–/ Ų/frame) was used with an exposure time of 9 s (200 ms/frame) and defocus range of −1.5 μm to −2.5 μm. For the +Ca²⁺datasets B and C (Supplementary file 1), micrographs were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a K2 Summit direct electron detector with a calibrated pixel size of 1.07325 Å/pixel. A total dose of 69.97 e^–/Ų distributed over 50 frames (1.39 e^–/ Ų/frame) was used with an exposure time of 10 s (200 ms/frame) and a defocus range of −1.5 μm to −2.5 μm. For all samples, automated data collection was carried out using Leginon (Suloway et al., 2005 (link)).

Data acquisition of human respiratory cilia microtubules for single-particle analysis was performed at the Harvard Cryo-EM Center for Structural Biology. Images were acquired on a Titan Krios microscope (Thermo Fisher Scientific) equipped with a BioQuantum K3 imaging filter (slit width, 25 eV) and a K3 detector (Gatan). Images were recorded at a defocus range of −0.8 to −2 μm with a nominal magnification of 64,000× in counting mode, resulting in a pixel size of 1.37 Å. Each image was dose fractionated into 50 movie frames with a total exposure time of 3.76 s and a total electron dose of ∼60 electrons per square Angstrom. Images were collected in 9 holes per stage move with 3 shots per hole. A total of 37,071 movies were collected using SerialEM version 3.8.6 (43 (link)) from 2 independent data collection sessions. An example micrograph is provided in Fig. 1.