Gatan 626 dh

Cryo-TEM was performed as described earlier (Rhiel et al. 2013 (link)). Briefly, a small droplet (5 μl) of the proteorhodopsin fraction, i.e., band B3 was placed on a copper grid covered by a holey carbon film (Quantifoil R 1.2/1.3, pore size 1.2 μm, 400 mesh; Quantifoil Micro Tools, Jena, Germany). Excess liquid was blotted for 3 s between two strips of filter paper. Subsequently, the sample was rapidly plunged into liquid ethane (cooled to about − 180 °C with liquid nitrogen) in a Cryobox (Zeiss, Oberkochen, Germany). The frozen specimen was transferred with a cryo-holder (Gatan 626-DH, Gatan, Pleasanton, USA) into a precooled cryo-transmission electron microscope (CM 120, FEI, Eindhoven, Netherlands) operated at 120 kV and viewed under low dose conditions. Cryo-TEM images were recorded with a 2 K CMOS Camera (F216, software EMMENU V4.0; camera and software TVIPS, Munich, Germany).

Freeze fracturing of liposomes incubated with proteins (2 µM) was done as described (Beetz et al., 2013 (link)). Cryo-TEM of liposomes coated with GST-syndapin III and syndapin III and of control incubations was performed on holey carbon film-covered copper grids. Liquid ethane-frozen samples (~−180°C) were transferred into a pre-cooled cryo-transmission electron microscope operated at 120 kV (Philips CM 120) using a cryo-transfer unit (Gatan 626-DH). Images were recorded with a 1K CCD Camera (FastScan F114, TVIPS).
Freeze-fracture replica were viewed using a Zeiss EM 902A transmission electron microscope run at 80 kV (Zeiss). Images were recorded digitally using an 1 k FastScan-CCD-camera (TVIPS camera and software).
Tubule diameters were analyzed using ImageJ. For diameter distribution analyses, measured diameters were grouped in 5 nm-step categories (0–5 nm,>5 to 10 nm,>10 to 15 nm and so on).