tilt series

MRC

57

(3838, 3710)

32 BIT FLOAT

(2.73, 2.73)

Cryo-electron tomography (cryo-ET) sample on the tubulation reaction mediated by SNX1-SNX5 in the presence of CI-MPR2330-2491. The CI-MPR-SNX1-SNX5 preincubated sample was added to 600 µM of liposomes in in buffer G. Before sample vitrification, BSA gold tracers (6nm) (Electron Microscopy Sciences) were added to the tubulation reaction in a 1:8 fiducials:reaction volume ratio. Four microliters of sample were incubated on a fresh glow-discharge grid for 30 s and blotted with filter paper before plunge-freezing in liquid ethane. Sample vitrification was performed on Vitrobot Mark II (FEI Company, USA) at 8°C and with a relative humidity close to saturation (90%). High-resolution cryoET dataset was obtained in an FEI Titan Krios G3 microscope, coupled with Gatan K2 Summit direct detector operated by Serial-EM software, at the cryoEM facility of Leicester University. Fifty-nine tomograms were acquired using a dose-symmetric scheme54 with tilt range ± 60⁰, 3⁰ angular increment and defocus values between -2 and -5 µm. For each tilt angle 10 frames were recorded with a dose rate of 0.29 e- /Å2 /frame and a total tomogram dose of 120 e- /Å2. Acquisition magnification was 53,000× rendering a pixel size of 2.73 Å.

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