Mark 4

For these studies, 8.5 µl of the aqueous water solution containing CPPs were blotted onto Quantifoil R2/2 grids (Quantifoil, Germany) by means of a Vitrobot Mark IV. Samples were vitrified utilizing liquid ethane. After blotting and plunge freezing samples were transferred into a Gatan cryo-stage and maintained at liquid Nitrogen temperature until transferred to the TEM utilizing a Gatan 626 cryo-holder.

After the final protein purification step by SEC, 3 µL of the 4F2hc-LAT2 sample at ≈3 mg/mL in SEC-buffer were adsorbed on glow discharged (20 s, 10 mA, 0.25 mbar) cryo-EM grids (Cu-R1.2/1.3 grids 300 Mesh; Quantifoil, Germany) for 3 s. The grids were plunge frozen in liquid ethane after blotting off excess buffer for 3 s using a Vitrobot Mark IV apparatus operated at approximately 100% humidity and cooled to 4–5 °C. The grids were stored in liquid nitrogen until further use. Cryo-EM data were collected using a 300 kV Thermo Fisher Scientific Titan Krios G4 electron microscope equipped with a Falcon 4 direct electron detector in an aberration-free image shifting and fringe-free illumination mode. The data were collected at a magnification of 96,000× corresponding to 0.83 Å/pix on the camera and at defocus ranges of −0.9 to −2.2 µm. Images were recorded in an automated fashion using the EPU 2 software (Thermo Fisher Scientific, Hillsboro, OR, USA) in counting mode for 4.6 s with a dose rate of 8.7 e⁻/Ų/s, resulting in a total accumulated dose on the specimen level of approximately 40 e⁻/Ų per exposure.

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)].