Cryo-EM Structural Analysis of GroEL-PepQ

PepQ was denatured in 8 M urea, 25 mM glycine phosphate, at pH 2 and incubated for 30 min at room temperature. Denatured PepQ (50 μM), in droplets of 4.6 μl (2.3 μM per addition) was titrated into solutions of either GroEL or Δ526 (8 μM tetradecamers, 100 μl) in TKM buffer, followed by rapid, repeated mixing and then incubated at room temperature for 5 min. The final concentration of PepQ was 7 μM. 3 μl of this PepQ/GroEL mixture was applied to a C-Flat 1.2/1.3 400 mesh holey carbon grid at 20 °C with 100% relative humidity and vitrified using a Vitrobot (Mark III, FEI company, Netherlands). The thin-ice areas that showed clear and mono-dispersed particles were imaged under an FEI Tecnai F20 electron microscope with a field emission gun (FEI company, Netherlands) operated at 200 KV. Data were collected on a Gatan K2 Summit direct detection camera (Gatan, Pleasanton CA) in electron counting mode70 (link) at a nominal magnification of × 19,000, yielding a pixel size of 1.85 Å. The dose rate was 10 e⁻ pixel⁻² s⁻¹ at the camera. A 33-frame movie stack was recorded for each micrograph, for a total exposure time of 6.6 s. The total dose onto the specimen was 19 e⁻ Å⁻².