Vitrobot marked 4

For cryo-EM analysis, 4 μl of D53 protein and D53−SKP1−D3−D14 complex at a concentration of 0.5–1 mg ml⁻¹ were applied to glow-discharged (HARRICK PLASMA, Ithaca, NY) holey carbon grids (Quantifoil Cu 1.2/1.3, 400 mesh) and then blotted and flash-frozen by FEI Vitrobot Marked IV. Cryo-EM data were collected in super-resolution mode with the defocus values varied from −0.7 to −0.9 μm following the methods in a previous report (Wang et al. 2019 ).

The Sr35–AvrSr35 complex grids were prepared for cryo-EM analysis. Holy carbon grids (Quantifoil Au 1.2/1.3, 300 mesh) were glow-discharged for 30 s at medium level in HarrickPlasma after 2 min evacuation. The purified Sr35–AvrSr35 protein was concentrated to approximately 0.5 mg ml^–1 and 3 µl of sample were applied to the grid. The grids were blotted for 2–3 s using a pair of filter papers (55 mm, Ted Pella Inc.) at 8 °C with 100% humidity and flash-frozen in liquid ethane using a FEI Vitrobot Marked IV. Stacks of Sr35–AvrSr35 cryo-EM samples were collected by a Titan Krios microscope operated at 300 kV, equipped with a K3 Summit direct electron detection camera (Gatan) using EPU 2 (Thermo Fisher Scientific, 2.8.1.10REL) at Zhengzhou University. Micrographs were recorded at 81,000× magnification corresponding to 1.1 Å per pixel. The defocus ranged from −1.5 µm to −2.0 µm. Each image stack contains 32 frames recorded every 0.11 s for an accumulated dose of approximately 50 e− per Ų and a total exposure time of 3.5 s. A second dataset from an independent protein purification was recorded at EMBL Heidelberg with the following parameters: Titan Krios microscope operated at 300 kV, equipped with a K3 Summit direct electron detection camera (Gatan), 50 e per Ų, 40 frames per stack.