Cryo-EM Sample Preparation for TsFpn-Fab Nanodiscs

The cryo grids were prepared using Thermo Fisher Vitrobot Mark IV. The Quantifoil R1.2/1.3 Cu grids were glow-discharged with air for 15 s at 10 mM in a Plasma Cleaner (PELCO EasiGlow^TM). Aliquots of 3.5 µl purified TsFpn-Fab in nanodisc were applied to glow-discharged grids. After being blotted with filter paper (Ted Pella, Inc.) for 4.0 s, the grids were plunged into liquid ethane cooled with liquid nitrogen. A total of 1838 micrograph stacks were collected for TsFpn-Fab and 1381 for TsFpn-Fab-hepcidin with SerialEM^{41 (link)} on a Titan Krios at 300 kV equipped with a K2 Summit direct electron detector (Gatan), a Quantum energy filter (Gatan) and a Cs corrector (Thermo Fisher), at a nominal magnification of 105,000× and defocus values from −2.0 µm to −1.2 µm. Each stack was exposed in the super-resolution mode for 5.6 s with an exposing time of 0.175 s per frame, resulting in 32 frames per stack. The total dose rate was about 50 e⁻/Ų for each stack. The stacks were motion corrected with MotionCor2^{42 (link)} and binned 2 fold, resulting in a pixel size of 1.114 Å/pixel. In the meantime, dose weighting was performed^{43 (link)}. The defocus values were estimated with Gctf^{44 (link)}.