Titan krios electron microscope

UltrAuFoil R1.2/1.3 300-mesh grids were glow discharged at 40 mA for 5 min using a GloQube glow discharge system (Quorum) and coated with graphene oxide flakes (Sigma). Next, 4 μl of 40 ng μl^–1 DDK was applied to each grid for 30 s before double-sided blotting for 4 s and plunge-freezing in liquid ethane using a Vitrobot Mark IV (FEI) operated at room temperature with 90% humidity. A total of 9,469 videos were collected at ×165,000 magnification (yielding a pixel size of 0.84 Å at specimen level) on a Titan Krios electron microscope equipped with a K2 Summit direct electron detector (Gatan Inc.).
A total of 2,967,226 particles were picked from motion-corrected video sums using crYOLO^{65 (link)}. CTF was estimated using Gctf^{64 (link)}, and 2D classification was performed after extraction of particles in a 240-pixel box in RELION-3.0.7 (ref. ³⁹ ).

For the AcrABZ-TolC/Puromycin sample, a 2.0 μl aliquot at 2 mg/ml was applied onto holey carbon film supported by a 200-mesh R2/1 Quantifoil grid (Quantifoil) that had been previously washed and glow discharged. The grid was blotted and rapidly frozen in liquid ethane using a Vitrobot IV (FEI) with constant temperature and humidity. The grid was stored in liquid nitrogen before imaging. Images of frozen-hydrated AcrABZ-TolC/puromycin particles were acquired on a FEI Tecnai G2 Polara electron microscope (FEI) operated at 300 kV using a K2 Summit direct electron detector camera (Gatan).
For the samples AcrABZ-TolC/MBX3132 and apo AcrAB-TolC, a 3 μl aliquot at a concentration of 2 mg ml⁻¹ was applied onto glow-discharged holey carbon grid (Quantifoil Au R1.2/1.3, 300 mesh). The grid was blotted and flash frozen in liquid ethane using a Vitrobot IV (FEI) with constant temperature and humidity. The grid was stored in liquid nitrogen before imaging. Zero-energy-loss images of frozen-hydrated AcrABZ-TolC/MBX3132 or apo AcrAB-TolC particles were recorded automatically on an FEI Titan Krios electron microscope at 300 kV, using a slit width of 20 eV on a GIF Quantum energy filter and a Gatan K2-Summit direct electron detector.
The data collection parameters for all three specimens are summarized in Supplementary file 1.

For the structure determination of saposin A discs with AcrB and AcrBZ as well as both complexes with supplementary cardiolipin, a 4.0 μl aliquot at 2 mg/ml was applied onto holey carbon film supported by a 300-mesh R1.2/1.3 Quantifoil gold grid (Quantifoil) that had been previously glow discharged. The grid was blotted for 2.5 – 3.0 seconds and rapidly frozen in liquid ethane using a Vitrobot IV (FEI) at 4°C and 100% humidity. The grids were stored in liquid nitrogen before imaging. Zero-energy-loss images of frozen-hydrated AcrB disc particles were recorded automatically on an FEI Titan Krios electron microscope at 300 kV, using a slit width of 20 eV on a GIF Quantum energy filter and a Gatan K2-Summit direct electron detector (Gatan) in counting mode. Images of frozen-hydrated AcrB/nanodisc-cardiolipin, AcrBZ/nanodisc and AcrBZ/nanodisc-cardiolipin particles were acquired automatically on the FEI Titan Krios electron microscope at 300 kV using a Falcon III direct electron detector camera (FEI) in counting mode.