replica data for manuscript—Cryo-electron tomography pipeline for plasma membranes—Nature Communications

The creation of platinum replicas on an EM grid was carried out as follows: unroofed cells on EM grids were fixed in 2% paraformaldehyde for 20 minutes, then in 2% glutaraldehyde at 4 °C overnight. After fixation, grids were incubated in 0.1% tannic acid at 4 °C for 20 minutes, washed with cold ddH2O, and incubated in 0.1% uranyl acetate at 4 °C for 20 minutes. Subsequent washing and stepwise dehydration in ethanol (15% to 100%, repeated three times) were done at room temperature, 4 minutes per step. EM grids were then critical-point dried and coated in a Leica EM ACE 900, with targets of 3 nm for platinum and 2.5 nm for carbon thickness. Coated grids were directly imaged using a 120 kV FEI Tecnai T12 with a Rio 9 CMOS camera. Montages at 5.87 Å/pixel resolution were collected using SerialEM, and blending was performed with the IMOD software suite. We evaluated plasma membrane-grid surface interactions by segmenting and quantifying clathrin structures using Mask R-CNN, categorizing them as flat, domed, or spherical based on curvature and lattice visibility.