Titan krios g3i electron microscope

For cryo-EM grid preparation, 3 µL of purified OTR:OT:G_o/q:scFv16 complex in blotting buffer were applied to glow-discharged holey carbon gold grids (Quantifoil R1.2/1.3, 300 mesh), and subsequently vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 100% humidity and 4 °C. Cryo-EM images were acquired by a Titan Krios G3i electron microscope (Thermo Fisher Scientific), operated at 300 kV, at a nominal magnification of 130,000 using a K3 direct electron detector (Gatan) in super-resolution mode, corresponding to a pixel size of 0.325 Å. A BioQuantum energy filter (Gatan) was operated in a zero-loss mode, using 20 eV energy slit-width. A total of 6,450 movies were obtained, with a defocus range of −0.8 to −2.4 µm using automatic data acquisition with EPU software (version 2.5;Thermo Fisher Scientific). The total exposure time was 1.79 s with an accumulated dose of ~63.78 electrons/Ų and a total of 67 frames per micrograph. A second set of 5,217 image stacks were acquired with the same conditions and parameters.

Four microliters of protein solution (total protein concentration, 1 mg ml⁻¹) were applied to freshly glow-discharged QUANTIFOIL R2/1 300 copper mesh grids (Quantifoil Micro Tools) and blotted for 5 s with a blot force of 5 at ~90% humidity and 8 °C using a Vitrobot Mark IV (Thermo Fisher Scientific) that was placed inside an anaerobic COY tent. For CHAPSO detergent-supplemented grids, 1 µl of detergent (dissolved in the same buffer as the protein) was added to a final concentration of 0.4% (m/V) to 3 µl of protein solution on the respective grid. Grids were plunge frozen in a liquid ethane (37 vol%) and propane (63 vol%) mix and stored in liquid nitrogen until data collection. CHAPSO-supplemented grids of AnfDGK were prepared to prevent preferential orientation.
Data of cryo-EM samples were collected on a Titan Krios G3i electron microscope (Thermo Fisher Scientific), operated at an acceleration voltage of 300 kV and equipped with a BioQuantum K3 energy filter (Gatan). Data were collected in electron counting mode at a nominal magnification of ×105,000 (0.837 Å per pixel) with a total dose of 50 e−/Ų (50 fractions), using the aberration-free image-shift correction in EPU v2.9–2.11 software (Thermo Fisher Scientific). The nominal defocus range used for data collection was −1.4 μm to −2.4 μm.

Translation initiation complex was programmed using 2 μM P. aeruginosa 70 S ribosome, 25 μM mRNA, 10 μM fMet-tRNA_i^fMet and incubated in final 70 S buffer (10 mM Hepes 7.6, 60 mM NH₄Cl, 15 mM KCl, 10 mM MgCl₂, 1 mM β-mercaptoethanol) at 37 °C for 15 min. The ternary complex was then incubated with 30 μM IF2 and 2 mM GDPCP (Millipore Sigma) at room temperature for 15 min. The mixture containing 2 μM P. aeruginosa 70S-IC (4 μl) was applied to Quantifoil R2/1 gold 200 mesh grids (Electron Microscopy Sciences) which were pre-cleaned in a Solarus 950 plasma cleaner (Gatan). The grids were plunged-frozen in liquid nitrogen-cooled ethane using a Leica EM GP2 cryo-plunger. Grids were transferred into a Titan Krios G3i electron microscope (ThermoFisher Scientific) operating at 300 keV and equipped with a GIF Quantum LS energy filter (Gatan) and a K3 direct electron detector camera (Gatan). The image stacks (movies) were acquired in super-resolution mode with pixel size of 0.425 Å/pixel. Data collection was done in the EPU software (ThermoFisher Scientific) setup to record movies with 31 fractions with a total accumulated dose of ~31 e^-/Ų/movie. A total of 8056 image stacks were collected with a defocus ranging between –0.2 and –2.1 µm. The statistics of data acquisition are summarized in Supplementary Table 1.

For this, 6 mg/ml of purified endogenous rat liver 20S proteasome was incubated on ice for 1 h with CBR3 at 1:50 molar ratio. Then, 2.5 µl of this mixture was applied to C-flat 2/2 300 mesh holey carbon grids (Protochips). Grids were blotted for 3 s at 4 °C and 100% humidity and plunge frozen to liquid ethane cooled by liquid nitrogen using a Vitrobot automated plunger (Thermo Fisher Scientific). The sample was applied to the grids 30 min after glow discharge to improve the percentage of side views. Titan Krios G3i electron microscope (Thermo Fisher Scientific) operating at 300 kV was used for the imaging at a nominal magnification of ×47,000, corresponding to a pixel size of 1.7 Å. A total of 596 movies were recorded on a Falcon 3EC direct electron detector (Thermo Fisher Scientific) using automated acquisition in EPU (v 2.5) software (Thermo Fisher Scientific). A nominal defocus range of −1.0 to −2.0 µm was used to collect the movies, and each movie was fractionated into 20 frames. The dose rate was set to ~0.96 e^-/pixel/s, and the total exposure time was 60 s, corresponding to an accumulated dose of ~20 e^-/Ų.

Grids preparation and data collection were carried out in S²C² (Menlo Park, CA). The WT MelB_St/Nb725_4/NabFab/anti-NabFab Nb complex was thawed out from –80°C storage. Aliquots of 3 μL of samples at 0.75 mg/mL or 1.5 mg/mL protein concentration were placed on 300-mesh Cu holey carbon grids (Quantifoil, R1.2/1.3) after glow-discharge (PELCO easiGlow) at 15 mA for 30 s and blotted on two sides for 3 s at 4°C and 100% humidity before vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). The grids were subsequently transferred to a Titan Krios (G3i) electron microscope (Thermo Fisher) operating at 300 kV and equipped with K3 direct electron detector (Gatan) and BioQuantum energy filter. The best grids were found from the sample at 1.5 mg/mL concentration.
A total of 14,094 and 8715 movies were automatically collected using the EPU Data Acquisition Software (Thermo Fisher) for non-tilted and 30° tilted collections, respectively. Both datasets were collected at a 0.86 Å pixel size and a dose of 50 e⁻/Å⁻² across 40 frames (0.0535 or 0.07025 s per frame for the non-tilted and tilted data collection, respectively) at a dose rate of approximately 23.36 or 17.8 e⁻/Ų/s, respectively. A set of defocus values was applied ranging from −0.8, to 1.0, −1.2,–1.4, or –1.8 μm with an energy filter slit width of 20 eV and a 100 μm objective aperture.

Translation initiation complex was programmed using 2 μM P. aeruginosa 70 S ribosome, 25 μM mRNA, 10 μM fMet-tRNA_i^fMet and incubated in final 70 S buffer (10 mM Hepes 7.6, 60 mM NH₄Cl, 15 mM KCl, 10 mM MgCl₂, 1 mM β-mercaptoethanol) at 37 °C for 15 min. The ternary complex was then incubated with 30 μM IF2 and 2 mM GDPCP (Millipore Sigma) at room temperature for 15 min. The mixture containing 2 μM P. aeruginosa 70S-IC (4 μl) was applied to Quantifoil R2/1 gold 200 mesh grids (Electron Microscopy Sciences) which were pre-cleaned in a Solarus 950 plasma cleaner (Gatan). The grids were plunged-frozen in liquid nitrogen-cooled ethane using a Leica EM GP2 cryo-plunger. Grids were transferred into a Titan Krios G3i electron microscope (ThermoFisher Scientific) operating at 300 keV and equipped with a GIF Quantum LS energy filter (Gatan) and a K3 direct electron detector camera (Gatan). The image stacks (movies) were acquired in super-resolution mode with pixel size of 0.425 Å/pixel. Data collection was done in the EPU software (ThermoFisher Scientific) setup to record movies with 31 fractions with a total accumulated dose of ~31 e^-/Ų/movie. A total of 8056 image stacks were collected with a defocus ranging between –0.2 and –2.1 µm. The statistics of data acquisition are summarized in Supplementary Table 1.