300 kv titan krios electron microscope

Data collection included 6811 images for the gpJ713, 2566 images for the gpJ713 and LamB complex, and 7714 images for the tail and LamB complex. Imaging of gpJ713-LamB complex and tail-LamB complex was performed using a 300 kV Titan Krios electron microscope (Thermo Fisher) outfitted with a K3 Summit counting camera (Gatan), with a pixel size of 1.0742. Imaging of gpJ713 was performed using a 300 kV Titan Krios electron microscope (Thermo Fisher) outfitted with a Falcon4 camera (Thermo Fisher), with a pixel size of 1.036. Each image stack received an approximate total dose of 50 e−/Ų, with defocus values ranging between −1.5 and −2.5 µm. Data collection was fully automated, facilitated by AutoEMation software^{35 (link)} and EPU (Thermo Fisher).

Best grids were used for small data set collection at a JEOL JEM-2200FS microscope, equipped with a K3 camera (Gatan). The data set analysis was useful to determine particle homogeneity and heterodimer integrity. Large data collection was performed at a high-end Titan Krios (Thermo Fisher Scientific) 300 kV microscope (Leicester, UK) equipped with a K3 camera (Gatan) and operated remotely. For this, the peak fraction of the size exclusion chromatography was applied to holey grids at concentrations between 1.2 and 1.5 mg ml⁻¹, vitrified and observed using a 300 kV Titan Krios electron microscope (Thermo Scientific™) equipped with a K3 camera. A total of 31,696 movies (50 frames) were collected in super-resolution mode using EPU. The set up used a 130 K magnification, which corresponded to 0.656 Å per pixel. The applied defocus covered a range from −0.8 to −2.4 μm, and the total electron dose was of 50 e⁻ Å⁻².

For cryo-EM sample preparation, three microliters of each protein were placed on glow-discharged holey carbon grids (Quantifoil Au R2.1/3.1, 300 mesh). The grid was blotted with filter paper for 3.5 s in a chamber set with 100% humidity at 8 °C to remove the excess sample and then plunge-frozen in liquid ethane cooled by liquid nitrogen with the Vitrobot Mark IV system (ThermoFisher Scientific). Cryo-EM specimens were imaged on a 300-kV Titan Krios electron microscope (ThermoFisher Scientific) using a normal magnification of 81,000 rpm. Movies were recorded using a Gatan K3 detector equipped with a GIF Quantum energy filter (slit width 20 eV) at the super-resolution mode, with a physical pixel size of 1.087 Å. Each stack of 32 frames was exposed for 2.56 s, with a dose rate of ∼23 counts/second/physical-pixel (∼19.5 e-/second/Ų) for each frame using EPU (ThermoFisher Scientific). All 32 frames in each stack were aligned and summed using the whole-image motion correction program MotionCor2^{60 (link)} and binned to a pixel size of 1.087 Å. The defocus value for each image varied from −1.5 to −2.0 μm and was determined by Gctf^{61 (link)}.