Purified substrate-bound Bam complex as described above was applied to glow-discharged Quantifoil R 2/1 holey carbon 400-mesh copper grids (Quantifoil). Grids were blotted for 4–5 s at 100% humidity with the blot force set to 16, and flash frozen by liquid nitrogen-cooled liquid ethane using a Thermo Fisher Scientific Vitrobot Mark IV (Thermo Fisher Scientific). The grid was then loaded onto a Titan Krios G3i electron cryo-microscope (Thermo Fisher) operated at 300 kV accelerating voltage. Image stacks (movies) were recorded on a Gatan Bioquantum K3 Imaging Filter (Gatan, USA) using the super-resolution counting mode and the calibrated magnification of 58717× using SerialEM49 . The slit of the energy filter was set to 25 eV, with a defocus range between 1.1 and 2.8 μm. The electron dose rate was 17 e−/physical pixel per second, and the subframe time was set to 0.06 second. A total exposure time of 3 second resulted in 50 subframes per image stack. The total electron dose was 70 e−/Å2 (~1.4 e−/Å2 per subframe). The multi-shot scheme in SerialEM was used for data collection, with settings of 4 holes per stage move, and 5 shots per hole, which greatly sped up the data collection. The data collection for both structures (4.1 Å and 6.5 Å) was performed in the same manner.
Titan krios g3i electron cryo microscope
Manufactured by Thermo Fisher Scientific
The Titan Krios G3i is a high-performance electron cryo-microscope designed for advanced structural biology research. It features a stable, high-resolution imaging system and advanced cryogenic capabilities to enable the study of biological samples at the nanoscale level.
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2 protocols using titan krios g3i electron cryo microscope
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Cryogenic Imaging of Bam Complex
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Cryogenic Imaging of Bam Complex
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Purified substrate-bound Bam complex as described above was applied to glow-discharged Quantifoil R 2/1 holey carbon 400-mesh copper grids (Quantifoil). Grids were blotted for 4–5 s at 100% humidity with the blot force set to 16, and flash frozen by liquid nitrogen-cooled liquid ethane using a Thermo Fisher Scientific Vitrobot Mark IV (Thermo Fisher Scientific). The grid was then loaded onto a Titan Krios G3i electron cryo-microscope (Thermo Fisher) operated at 300 kV accelerating voltage. Image stacks (movies) were recorded on a Gatan Bioquantum K3 Imaging Filter (Gatan, USA) using the super-resolution counting mode and the calibrated magnification of 58717× using SerialEM49 . The slit of the energy filter was set to 25 eV, with a defocus range between 1.1 and 2.8 μm. The electron dose rate was 17 e−/physical pixel per second, and the subframe time was set to 0.06 second. A total exposure time of 3 second resulted in 50 subframes per image stack. The total electron dose was 70 e−/Å2 (~1.4 e−/Å2 per subframe). The multi-shot scheme in SerialEM was used for data collection, with settings of 4 holes per stage move, and 5 shots per hole, which greatly sped up the data collection. The data collection for both structures (4.1 Å and 6.5 Å) was performed in the same manner.
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