Cf 2 1 3c

Gold fiducial markers (10 or 5 nm) (BBI Solutions) in buffer A were added to the tubulation reaction (1:10 fiducials:reaction volume ratio). Four microliters of this mixture was backside-blotted for 6 s at a relative humidity of 98% and a temperature of 19°C on a glow-discharged holey carbon grid (CF-2/1-3C; Protochips) before plunge-freezing in liquid ethane (Leica EM GP2 automatic plunger). Dose-symmetrical tilt series acquisition (55 (link)) was performed on an FEI Titan Krios electron microscope operated at 300 kV using a Gatan Quantum energy filter with a slit width of 20 eV and a K2 or K3 direct detector operated in counting mode. The total exposure of ~130 e⁻/Ų was equally distributed between 41 tilts. Ten frame movies were acquired for each tilt. The details of data collection are given in table S1. The selection of acquisition areas was guided by suitability for high-resolution tomographic data collection (i.e., vitreous ice quality, lack of crystalline ice contaminations, and intactness of the carbon support) and was not based on the morphology of tubules.

AP2 was recruited to liposomes alone or in the presence of the FCHO2 linker by mixing 1.5 μM AP2 or 1.5 μM AP2 and 7.5 μM FCHO2 linker, with 100-nm extruded liposomes (0.2 mg/ml) containing 10% brain PtdIns(4,5)P₂ and 10% DOPS in a POPC/POPE (3:2) mixture in HKT buffer. Reactions were performed in parallel and incubated for 30 min at 21°C. After incubation, the reactions were supplemented with 1:10 of 10-nm gold fiducial markers in HKT buffer. A total of 3.5 μl of this mixture was applied on a glow-discharged holey carbon grid (CF-2/1-3C, Protochips) and back-side blotted for 4 s at relative humidity of 98% and 18°C, followed by plunge-freezing in liquid ethane (Leica EM GP2 automatic plunger).
Data acquisition was performed as in (14 (link)). Dose-symmetrical tomographic tilt series (84 (link)) were collected in a FEI Titan Krios electron microscope operated at 300 kV with a Gatan Quantum energy filter with a slit width of 20 eV and a K3 direct detector operated in counting mode using tilt series controller in Serial EM software. Tilt series contained 41 tilted images (−60° to +60° with 3° increment) with 10-frame movies acquired for each tilt and were imaged with a total exposure of ~130 e⁻/A² equally distributed between tilts. The details of data collection are given in table S1.