Fei vitrobot mark 4

For the MSP1E3D1 nanodisc samples, 100 µM of DCPIB (Tocris, Bristol, UK) was added to sample to give a final concentration of 0.8 mg/mL LRRC8A-MSP1E3D1. DCPIB was allowed to equilibrate and bind complex on ice for 1 hr prior to freezing grids. Sample with drug was cleared by a 5 min 21,000 x g spin prior to grid making. For freezing grids, a 3 µl drop of protein was applied to freshly glow discharged Holey Carbon, 400 mesh R 1.2/1.3 gold grids (Quantifoil, Großlöbichau, Germany). A FEI Vitrobot Mark IV (ThermoFisher Scientific) was utilized with 22°C, 100% humidity, one blot force, and a 3 s blot time, before plunge freezing in liquid ethane. Grids were then clipped in autoloader cartridges (FEI, Hillsboro, Oregon) and shipped in a dry shipper for data collection. The MSP2N2 sample was frozen without drug at 0.8 mg/mL with the same conditions as the MSP1E3D1 grids.
For the digitonin sample, purified protein was shipped in a refrigerated container. The next day, for freezing, a 3 µL drop of protein was applied to a freshly glow discharged Holey Carbon, 400 mesh R 1.2/1.3 gold grid (Quantifoil). A FEI Vitrobot Mark IV (ThermoFisher Scientific) was utilized with 22°C, 100% humidity, one blot force, and a 5 s blot time, before plunge freezing in liquid ethane. Grids were then clipped and used for data collection.

Freshly assembled preparations of the different complexes (1 μM) were used to prepare cryo-grids by applying 3.7 μL of complex to a holey-carbon grids (Quantifoil™ 1.2/1.3, 300 mesh copper) which was glow discharged in air with PELCO easiGlow by applying a current of 10mA for 60 s. Grids were blotted for 3 s at 25°C and plunged into liquid ethane using FEI-Vitrobot Mark IV (Thermo Fisher Scientific) or Leica EM GP2 (Leica microsystems) and stored in liquid nitrogen prior to imaging. Data were collected using FEI Titan Krios electron microscopes at 300kV, equipped with a Gatan BioQuantum (slit width 20eV) K2 direct electron detectors operating in counting mode. The R2-TTT and R2-T2 (TTi1-TTi2) data were collected at Astbury Biostructure Laboratory, Leeds, UK and R2TP-TTT data was acquired at eBIC Diamond Light Source, UK. Automated data acquisition software EPU (Thermo Fisher Scientific) was used to collect data based on a published protocol (29) and with the parameters described in Table S1. Micrograph movies were collected with 40 fractions across a 10 s exposure. Autofocusing was performed after every 10 μm. For R2-T2 and R2-TTT data was collected using total doses of 49.6 e^-/Ų and 51.2 e^-/Ų respectively and the R2TP-TTT data was acquired with dose of 42.12 e/Å ² over 40 fractions.

The cell wall peels previously incubated in HEPES buffer were laid on a slide with a drop of HEPES buffer to keep the cell wall hydrated. After incubation in HEPES buffer, the peels were mounted in a drop of HEPES on a slide. A tangential light was shined at the peel to increase visibility. If possible, a magnifying glass affixed on a support can be used. Small rectangular pieces (∼2 x 3 mm) were cut out of the cell wall peel with a sharp razor blade and carefully dragged on the carbonated side of glow-discharged (15mA – 1min) Quantifoil R2/2 NH2 Cu EM grids (EMSdiasum). Plunge freezing was performed with a 60/40 ratio ethane/propane mix and an FEI Vitrobot Mark IV (Thermo Fisher). Humidity was set at 50%, temperature at 20°C. Grids were first manually backblotted for 6 s in order to attach the cell wall peel firmly to the carbon, followed by two autoblottings (front and back) 5 s, maximal blot force (25) and a drain time of 3 s.