Gloqube plus

Peak fractions of freshly purified PIN8 were concentrated to 4–10 mg ml⁻¹. C-flat Holey Carbon grids (CF-1.2/1.3, Cu-300 mesh) were glow-discharged for 45 s at 15 mA in a GloQube Plus (Quorum). A drop of 4 µl of sample was applied to the non-carbon side of the grids, and blotted with a Vitrobot Mark IV (ThermoFisher Scientific) operating at 4 °C and 100% humidity and using blot time of 4 s, before plunge-freezing into liquid ethane. The substrate-bound states were obtained by incubating the sample with 15 mM of IAA sodium salt or 2 mM of NPA for 2 h prior to grid freezing.

LUVs of different lipid compositions were prepared from MLVs as described above. Experiments with LLO only were performed at different incubation times (30 min, 2 h and 6 h), at 20 °C or 37 °C and pH 6.5. For experiments with LLO and LmPC-PLC preincubation, all the incubation steps were performed at 37 °C. 2.5 mM LUVs were preincubated with 5 μM LmPC-PLC for 30 minutes. After that, LLO was added to a final concentration of 5 μM and the sample mixtures were further incubated for 60 minutes. After incubation, 3 μl of each sample was transferred to glow-discharged (GloQube® Plus, Quorum, UK) Quantifoil R1.2-1.3 grids (Quantifoil, Germany) and blotted under 100% humidity using Mark IV Vitrobot (Thermo Fisher Scientific). Blot force used was 3 and blot time was 6.5 s. Micrographs were collected on cryo-transmission electron microscope Glacios (Thermo Fisher Scientific) operated at 200 kV and equipped with Falcon 3 direct electron detector (Thermo Fisher Scientific), at a nominal magnification of 5,300x and 73,000x and defocus range of –3 µm to –4 µm.

C-flat™ 2/2, 200 mesh holey carbon grids (Protochips, Morrisville, NC, USA) were glow discharged: 20 mA, 60 s, positive polarity, air atmosphere (GloQube^® Plus, Quor-um, Laughton, UK). Then, 3 µL of the sample was applied to the grid, blotted, and vitrified in liquid ethane on Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). Vitrobot conditions were set to 100% relative humidity, 4 °C, blot force: 2 and blot time: 7 s. Samples were visualized under cryogenic conditions using a 200 kV Glacios microscope with a Fal-con 3EC detector (Thermo Fisher Scientific, Waltham, MA, USA).

Quantifoil grids were rendered hydrophilic using a glow discharge device (GloQube Plus, Quorum) for a time of 45 s in air before specimen application to the grid. An aliquot of 3.5 µl stock solution of TMV at a concentration of approximately 90 mg ml⁻¹ or 2.5 µM was applied onto a 200 mesh Quantifoil grid with regular R2/2 holey carbon support. The excess of the applied droplet was blotted using a Vitrobot Mark IV (Thermo Fisher Scientific MSD). Due to the high concentration, optimal results for obtaining thin ice across the 2 µm holes were achieved using a high blot force of +10 and a duration for blotting of 10 s, before plunge freezing. Grids were prepared at 4 °C and 100% humidity. To maximize occupancy of holes with rafts of TMV rods, application of TMV at high concentrations was critical. After flash freezing, grids were stored in grid boxes in liquid nitrogen for subsequent mounting into autogrids of the Krios G4 Cryo-Autoloader. For the KLH samples, cryo-grids were prepared as described above. For KLH, however, a 10 mg ml⁻¹ or 2.5 µM solution was plunge frozen using a blot force of −10 and 6 s blotting time.

Quantifoil^® R 2/2 (or 1.2/1.3), 200 (Quantifoil Micro Tools GmbH, Großlöbichau, Germany) EM grids were glow discharged for 60 s at 20 mA and positive polarity in air atmosphere (GloQube^® Plus, Quorum, Laughton, UK). Vitrobot conditions were set to 4 °C, 95% relative humidity, blot time: 5 s, and blot force: 4. An amount of 2 µL of the sample suspension was applied to the grid, blotted, and plunge-frozen in liquid ethane with Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). Samples were visualized under cryo conditions with a Falcon 3EC detector on a 200 kV microscope Glacios (Thermo Fisher Scientific, Waltham, MA, USA).

3.5 µL of the SecM-SRC sample (8 OD₂₆₀/ml) were applied to grids (Quantifoil, Cu, 300 mesh, R3/3 with 3 nm carbon, Product: C3-C18nCu30-01) which had been freshly glow discharged using a GloQube® Plus (Quorum Technologies) in negative charge at 25 mA for 30 s to make the grids hydrophilic. Sample vitrification was performed using mixture of ethane/propane in 1:2 ratio in a Vitrobot Mark IV (ThermoScientific), with the chamber set to 4 °C and 100% rel. humidity, and blotting performed for 3 sec with zero blot force with Whatman 597 blotting paper. The grids were subsequently clipped into autogrid cartridges and stored in liquid nitrogen until needed.

Samples of SCPs were prepared using Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). Quantifoil^®® R 2/2, 200 mesh holey carbon grids (Quantifoil Micro Tools GmbH, Großlöbichau, Germany) were glow-discharged for 60 s at 20 mA and positive polarity in the air (GloQube^®® Plus, Quorum, Laughton, UK) [55 (link)]. Conditions were set at 4 °C, 100% relative humidity, blot time: 5 s, and blot force: 4. An amount of 2 µL of the sample with SCPs in suspension was applied to the grid, blotted, and vitrified in liquid ethane. Samples were visualized under cryogenic conditions using a 200 kV microscope Glacios with Falcon 3EC detector (Thermo Fisher Scientific, Waltham, MA, USA). The total electron dose was 30 eA⁻².