Ultraufoil r 1.2 1 3 au 300 mesh gold grids

Proteasome was obtained as a gift from Yifan Cheng. UltrAuFoil R 1.2/1.3 Au 300 mesh gold grids (Quantifoil) were used for cryo-EM sample preparation. The grids were plasma treated with the Solarus Plasma Cleaner 950 (Gatan, 75% argon/25% oxygen atmosphere at 15 Watts for 30 s) immediately before sample application to make their surfaces hydrophilic. 3 uL of proteasome at 0.3 mg mL⁻¹ concentration was applied to grids and plunge-frozen using a Leica EM-grid plunger (Leica Microsystems), with the chamber humidity maintained at 90% humidity and 4 °C.

For data collected at Scripps Research, apoferritin was purchased (Thermo Fisher Scientific) at a concentration of approximately 8 µM (3.5–4.0 mg mL⁻¹). Two dilutions were prepared having final concentrations of ~1.6 and 0.8 µM in a buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl 0.5 mM TCEP. Untilted, 10° and 20° tilted datasets were collected with grids prepared from ~1.6 µM apoferritin and the rest of the datasets were collected with grids prepared from ~0.8 µM apoferritin.
UltrAuFoil R 1.2/1.3 Au 300 mesh gold grids (Quantifoil) were used for cryo-EM sample preparation. The grids were plasma treated with the Solarus Plasma Cleaner 950 (Gatan, 75% argon/ 25% oxygen atmosphere at 15 Watts for 7 s) immediately before sample application to make their surfaces hydrophilic. A 3 μL aliquot of apoferritin sample at ~1.6 or 0.8 µM concentration was applied to grids and then vitrified in liquid ethane using a manual plunger. All operations were performed in a 4 °C cold room at >80% humidity to minimize evaporation and sample degradation.
For data collected at Janelia Research Campus, apoferritin was purified as described before^{72 (link)}. Grids were vitrified in a manner similar to the grids prepared for data collection at Scripps Research.