Cryo-EM structure of recombinant TRAP1

Recombinant human TRAP1 was expressed in E. coli BL21 cells and purified as described previously [15 (link)]; 4 μM purified human TRAP1 was incubated with 1 mM AMPPNP and 1 mM MgCl₂ at 37 °C for 30 min before application to the grid (Quantifoil holey carbon grid, 400 mesh) and vitrified using a Vitrobot Mark IV. A total of 665 micrographs were collected on a Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV with a K2 Summit direct electron detector (Gatan, Inc.) and a slit width of 20 eV on a GIF-BioQuantum energy filter. Images were recorded with SerialEM with a super-resolution pixel size of 0.516 Å. Defocus varied from 0.6 to 2.2 μm. Each image was dose-fractionated to 60 frames (0.2 s each, total exposure of 12 s) with a dose rate of 6 e⁻/Ų/s for a total dose of 72 e⁻/Ų. Image stacks were motion-corrected and summed using MotionCor2 [76 (link)], resulting in Fourier-cropped summed images with 1.032 Å/pixel. CTFFIND4 was used to estimate defocus parameters for all the images [77 (link)]. Initial particle picking was carried out using Gautomatch without a template to generate the 2D class averages, which were then used as templates for a second-round particle picking on micrographs with 25 Å low-pass filtering. Two rounds of reference-free 2D classification were performed for 25 iterations each with images binned by 2 using Relion 3.0 [78 (link)].