Filament assembly, EM sample preparation, data acquisition and processing
To assemble DRP1-MID49126-454 filaments, the proteins were mixed to a final concentration of 2 μM each and kept for an hour at room temperature. The mixture was dialyzed against assembly buffer- 20 mM HEPES pH 7.5, 50 mM KCl, 3 mM MgCl2, 1 mM DTT and 200 μM GMPPCP with or without 0.2% octyl-glucopyranoside (Anatrace). The filaments were observed using negative stain TEM or cryoEM after vitrification. Under these conditions, the mutant DRP1S611D failed to coassemble with MID49, but upon further lowering the ionic strength to 25mM KCl, DRP1S611D displayed detectable but greatly reduced coassembly compared to WT protein. For vitrification, the sample was applied to Quantifoil holey carbon grids (R2/2) using a Vitrobot Mark III with 3.5 μl sample, 5 seconds blotting time and a 0 mm offset at 19°C and 100% humidity. Images were collected on an FEI T30 Polara operating at 300kV at a magnification of 31000X. Images were recorded on a Gatan K2 summit camera in super resolution mode for a final binned pixel size of 1.22 Å/pixel. The movies were dose fractionated, contained 30-40 frames, had a total exposure time of 6-8 seconds with 0.2 seconds per frame and a per-frame dose of 1.1 to 1.4 electrons/Å2. SerialEM was used to automate data collection55. The defocus range was 0.8-3 μm under focus. The data was motion corrected and dose-weighted using UCSF Motioncor256. CTF parameter estimation on the non-dose-weighted but motion-corrected stacks was done using CTFFIND4 and GCTF57,58.
Filaments were boxed using the program e2helixboxer.py from the EMAN2 suite59. Particle coordinates were used to extract discrete particles using RELION 1.3-1.460 and all further processing was done within the RELION suite. Multiple rounds of 2D classification identified the well-ordered segments. 3D autorefine was run using a customized Relion1.2 version with the IHRSR algorithm implemented61,62. The consensus helical structure was used to classify the particles without refining helical symmetry (using RELION 1.4), resulting in 2 major classes that differed slightly in rise and twist (Extended data figure 2c). Particles from each class were selected and independently refined again with helical RELION 1.2 and IHRSR. Analysis of these reconstructions revealed that each structure was comprised of three linear filaments that bundle together to form a structure that resembled a triangle in cross-section (Extended data figures 2–3). The vertices of the triangle are formed through asymmetric interactions between the G-domains in adjacent filaments. The triangular arrangement of the bundled helices is unlikely to correspond to a biologically meaningful architecture, and this structure cannot form if the MID49 receptor is embedded in the outer mitochondrial membrane.
To further improve signal-to-noise, each of the three filaments in each independent half-map was segmented, extracted, resampled on a common grid and summed using UCSF Chimera63–67. The respective symmetrized but unfiltered half maps from each class were again aligned to a common grid and summed according to the C2 symmetry axis of the DRP1 dimer. In a last step, relion_postprocess was used to add the resulting and fully symmetrized half maps (Extended data figure 2c). These half maps and the final summed map, with differential B-factor sharpening per region (Extended data figures 2c, 4–5), were used for atomic modeling using Rosetta as described below.
For the projection structure of the DRP1G362D rings, 2 μM protein was mixed 1:1 molar ratio with MID49126-454 and was allowed to sit at room temperature for an hour. The mixture was dialyzed against the assembly buffer (without detergent) overnight. The sample was collected after 12 hours and vitrified using ultra-thin 3 nm carbon support films (Ted Pella). For vitrification, a Mark III vitrobot was used with 3.5 μl sample, 0 mm offset, 100% humidity and 3.5 seconds blot time. The images were collected using an FEI TF20 microscope and SerialEM for automated data collection. The data were recorded with a Gatan K2 camera operating in super resolution mode to collect dose fractionated movie stacks with a final binned pixel size of 1.234 Å/pixel. 40 frames were collected per stack (0.2 seconds per frame and 1.42 electrons/Å2). The movie stacks were motion corrected and the parameters of the transfer function were estimated as described above. Approximately 2000 particles were picked manually for initial 2D classification in RELION 1.4 and these averages were used as templates for further particle picking by Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/). Final 2D averages of the entire rings versus quarter segments of the rings were computed using Relion1.4.