Titan krios cryo stem

Five μL of the assembled protein was pipetted onto glow-discharged Quantifoil holey carbon TEM grids (Electron Microscopy Sciences). Excess fluid was removed before the sample was flash frozen hydrated by plunging into a bath of liquid ethane slush using the FEI Vitrobot Mark IV [56 (link)]. The grids were stored in liquid nitrogen until imaged with the Tecnai G² F20, operated at an accelerating voltage of 200 kV or Titan Krios cryo-STEM (FEI) at an accelerating voltage of 300 kV. Images were recorded under low dose conditions with a Gatan Ultrascan 4000 CCD camera at a nominal magnification of 50,000 X corresponding to a pixel size of 0.22 nm and a defocus level ranging from − 1.5 to − 2.5 μm.

Tomograms of the GMP-PNP-bound DRP1 helices attached with Ni-NTA-Nanogold were acquired with the FEI Tecnai G² F20 cryo-STEM at 200 kV. Images were recorded at a nominal magnification of 25,000X corresponding to a pixel size of 0.48 nm (for the NanoVan-stained samples). The tomograms for DRP1 helices unbound to the Nanogold were collected with the FEI Titan Krios cryo-STEM at 300 kV using a tilt range between −60° to 60° at 18,300 fold magnification corresponding to a pixel size of 0.820 nm. The images were aligned, cropped, and binned using IMOD [61 (link)–63 (link)]. One representative tomogram (acquired in cryo condition), binned twice to 1.64 nm/pixel, was manually traced in 3dmod, Version 4.5 [60 (link)]. Chimera was used to compare the DRP1 model helices to the extracted helix data traced in 3dmod. Additionally, the pitches and diameters of the helices were manually measured on the digital images.