Vitrobot mark 4

For peptidisc-embedded MsbA, 3.5 μL aliquots at 1.3 mg/mL were applied to glow-discharged Quantifoil R2/2 400 mesh Cu holey carbon grids. Grids were blotted for 3.5 s at 4°C with ~90% humidity and, after a waiting time of 10 s, plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). For peptidisc-embedded MscS, 3.5 μL aliquots at 0.07–0.12 mg/mL were applied to glow-discharged Quantifoil R1.2/1.3 400 mesh Cu holey carbon grids covered with a homemade thin carbon film. Grids were blotted for 0.5 s at 4°C with 80–90% humidity and then plunge-frozen in liquid ethane using a Vitrobot Mark IV.
Cryo-EM data were collected using SerialEM on a 300-kV Titan Krios electron microscope (Thermo Fisher Scientific) in the Cryo-EM Resource Center at The Rockefeller University, equipped with a K2 Summit direct electron detector (Gatan) in super-resolution counting mode at a nominal magnification of 29,000x, corresponding to a calibrated super-resolution pixel size of 0.5 Å/pixel. Exposures of 10 s were dose-fractionated into 40 frames (250 ms per frame), with a dose rate of 8 electrons/pixel/s (~2 electrons/Ų/frame), resulting in a total dose of 80 electrons/Ų. The defocus range was varied from −1.0 to −2.0 μm for MsbA, and from −1.5 to −3.5 μm for MscS.

For the initial 7.2 Å map of pre-pore wt XptA2, unfrozen protein (at 6 mg/mL in 16 mM tris, 40 mM NaCl, 20% glycerol, pH 7.5) was shipped to the cryo-EM core operated by the Baylor University College of Medicine (BCM) for sample processing. Protein was diluted in glycerol-free tris buffer to 1.0 mg/mL at BCM. Grids were frozen using a Vitrobot Mark IV (ThermoFisher Scientific, Waltham, MA, USA) using blot time of 4 sec, force of 2, and plunged into liquid ethane. Grids were screened for adequate particle density and ice thickness using a JEOL2010F microscope.
For the high-resolution pre-pore structure of XptA2 and the structure of 2-fragment XptA2, cryo-EM grids were prepared using a Vitrobot Mark IV (ThermoFisher Scientific, Waltham, MA, USA). Four microliters of XptA2 wt at 1.5 mg/mL for each sample (10 mM Tris, 100 mM NaCl, pH 7.5) were applied to a glow-discharged Quantifoil holey carbon grid (R2/2), blotted for 4 s at a blot force of 4 before plunge freezing into liquid ethane. Grids were stored in liquid nitrogen before shipping for screening and data collection.

For sample vitrification, we firstly pipetted 7 μl sample buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl) onto the functionalized graphene grids, and incubated the grids for 2 min at a chamber of high humidity. Afterwards, the grids were edge-blotted by filter papers to remove the extra buffer, immediately followed by adding 4-μl sample solution, incubated for 2 min and then transferred into Vitrobot Mark IV (ThermoFisher Scientific). The humidity of Vitrobot chamber was set as 100%, and the temperature as 8 °C. The grids were blotted by filter papers (Ted Pella) for 1 s with a force of −2, and then plunge-frozen into liquid ethane. The vitrified specimens were stored in liquid nitrogen. To prepare conventional graphene-supported cryo-specimen, we pipetted 4 μl sample solution onto freshly glow-discharged graphene grid, and blotted the grid for 1~2 s with a force of −2, using Vitrobot Mark IV (ThermoFisher Scientific). After blotting, the grid was frozen into liquid ethane and stored in liquid nitrogen.

For the cryo-EM grid preparation, 3 μL of the purified 5-CT–5-HT_5A–G_i complex at a final concentration of 25 mg/mL was applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3, 300 mesh), and vitrified using a Vitrobot Mark IV (ThermoFisher Scientific) subsequently. Grids were plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fischer Scientific). Frozen grids were transferred to liquid nitrogen and stored for data acquisition. Cryo-EM images were collected by an FEI Titan Krios at 300 kV accelerating voltage equipped with a Gatan K3 Summit direct electron detector at the Center of Cryo-Electron Microscopy Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). A total of 5303 movies were automatically acquired using SerialEM10 in super-resolution counting mode at a pixel size of 1.071 Å. The images were recorded at a dose rate of about 26.7 e/Ų/s with a defocus ranging from –1.2 to –2.2 μm. The total exposure time was 3 s, and intermediate frames were recorded in 0.083-s intervals, resulting in a total of 36 frames per micrograph.

Protein for single particle analysis was prepared using Quantifoil R 1.2/1.3 Cu 300 grids, glow-discharged in air using the Quorum GloQube^® for 30 s at 40 mA (Vip3Bc1) or plasma cleaned using a Tergeo-EM plasma cleaner (Pie Scientific) (Vip3Bc1^act). 3 µL of sample was applied to the grids in a Vitrobot Mark IV (Thermo Fisher Scientific) at 95% relative humidity, 4 °C, blotted for 6 s with a blot force of ‘6’ and plunged into liquid ethane.
For grids containing LUVs, samples were vitrified ~6 h after LUV and protein were mixed. LUV solution was mixed 2:1 with a concentrated stock of 10 nm fiducial markers resuspended in the same buffer as LUVs. Quantifoil R 2/2 Cu 200 grids were glow-discharged in air in a Quorum GloQube^® for 30 s at 40 mA prior cryoEM grid preparation. 3 µL of sample was applied to the grids in a Vitrobot Mark IV (Thermo Fisher Scientific) at 95% relative humidity, 4 °C, blotted for 6 s with a blot force of ‘6’ and plunged into liquid ethane.