Structural Analysis of Crosslinked WVZ Complex

The purified crosslinked WVZ complex was applied to Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated with the gel filtration buffer. The complex was diluted to 35 ng/μL with SEC buffer. Five microliters of sample were applied to the glow-discharged 200 mesh carbon-coated copper grids. The samples were stained using 0.75% uranyl formate and air-dried. Data were collected on a Talos L120C transmission electron microscope equipped with a 4K × 4K CETA CCD camera (Thermo Fisher Scientific). Images were recorded at a nominal magnification of 92,000×, corresponding to a pixel size of 1.55 Å. CTF parameters of each micrograph were determined using CTFFIND4.^{39 (link)} Particles were picked and subjected to two rounds of 2D classification, followed by de novo 3D model generation, 3D classification, and auto-refinement in Relion.^{37 (link)}