Trimer (500 μg) was incubated with 10-fold molar excess of 43A2 Fab overnight. The complex was then purified over Superose 6 column (GE Healthcare), concentrated to 1.5 mg/ml, and mixed with Lauryl Maltose Neopentyl Glycol (Anatrace) before deposition onto 2/2 Quantifoil grids (EMS) that were glow-discharged for 10 s, directly preceding the deposition in a Vitrobot (Thermo Fisher Scientific). Once sample was deposited, the grids were blotted and plunged into liquid ethane using the Vitrobot to immobilize the particles in vitreous ice. Using Leginon image acquisition software, we collected 1366 micrographs at a nominal magnification of ×29,000 with a Gatan K2 summit detector mounted on a Titan Krios set to 300 kV set to counting mode for the data collection (44 (link)). The dose rate was ~4.78 e−/pix per second with frame exposure of 250 ms, with a total exposure time and dose of 14 s and 60 e−/Å2, respectively. MotionCor2 was used for frame alignment, and CTF models were determined using GCTF (49 (link)). DoG Picker was used to pick 455,207 particles, which were then extracted and subsequently 2D-classified in cryoSPARC (45 (link), 50 (link)). Selected 2D classes amounting to 85,841 particles were then fed into the 3D homogeneous refinement algorithm using C3 symmetry, resulting in final resolution of ~3.52 Å.
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