Cryo-EM Sample Preparation for CLPB Proteins

Cryo-grids were prepared with an FEI Vitrobot Mark IV with the chamber set at 6°C and 100% humidity. A total of 4 μL aliquots of the prepared samples described above were applied onto the glow-discharged holey-carbon grids (R1.2/1.3, Au, 300 mesh, Quantifoil) and blotted after a 30-s waiting time. The grids were then flash frozen using liquid ethane. The cryo-grids were screened using an FEI Talos Arctica microscope operated at an accelerating voltage of 200 kV, and the qualified ones were recovered for data collection. To address the issue of strong preferred orientation of the particles distribution in the cryo-grids for CLPB and CLPB^E425Q samples, 0.5 mM CHAPSO was added to these 2 samples, as described previously [53 (link)].