200 kv glacios microscope

For cryo-EM analysis, 3 μl of TarL H300N (9.3 mg/ml) with 5 mM CDP-ribitol in 20 mM Hepes (pH 7.5), 500 mM NaCl, and 6.5 mM CHAPS was applied to a glow-discharged Lacey carbon 300 mesh grid (Ted Pella, 01895-F), blotted for 1.5 s at 100% humidity, and plunge-frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Grids were screened at the High-Resolution Macromolecular Cryo-electron Microscopy (HRMEM) facility (Vancouver, British Columbia, Canada) on a 200-kV Glacios microscope (Thermo Fisher Scientific) equipped with a Falcon III camera (Thermo Fisher Scientific). A total of 19,380 movies were collected at the Pacific Northwest Center for Cryo-EM (PNCC; Portland, Oregon, USA) on a 300-kV Titan Krios (Thermo Fisher Scientific) equipped with a K3 camera (Gatan). Data were collected in super-resolution counting mode with 0.5295 Å per super-resolution pixel (1.059 Å per physical pixel; calibrated magnification of 81,000×). Each movie was collected with a total dose of 50 electrons/Ų fractionated across 50 frames. Automated data collection was carried out using SerialEM with a nominal defocus range set from −1 to −2.5 μm.

The protein complex was diluted to 0.5 mg/ml in the presence of 0.01% fluorinated octyl maltoside. Aliquots of 3 μl were applied to glow-discharged 300 mesh Quantifoil (1.2/1.3) grids, which were blotted for 2 s at 4°C and 100% humidity with an offset of −15 and plunged frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Grids were screened at the Stanford cEMc on a 200 kV Glacios microscope (Thermo Fisher) equipped with a K3 camera (Gatan). A dataset was collected on a single grid using a 300 kV FEI Titan Krios microscope located at the HHMI Janelia Research Campus. Movies were collected using a 6eV wide energy filter at 165,000× magnification, corresponding to physical pixel size of 0.743 Å. Automated data collection was carried out using SerialEM with a total dose of 60 e⁻/Ų in 42 frames over a nominal defocus range of −0.8 to −1.8 μm.

All constructs analysed by cryo‐EM were prepared in the following way: Open‐hole R1.2/1.3 grids (Quantifoil) with 200 mesh were used for plunge freezing. Grids were glow‐discharged with BalTec SCD 005 sputter coater for 120 s at 25 mA using residual air. Four microliters of the sample was applied onto the treated side and front‐side blotted using the Leica GP2 plunge freezer. UBR5 constructs were frozen in GF Buffer with the addition of 4 mM CHAPSO or 0.005% (w/v) fluorinated octyl β‐maltoside (Anatrace) prior to freezing.
Initial grid screening was performed on the 200 kV Glacios microscope (ThermoFisher) using the Falcon III detector (ThermoFisher). For high‐resolution structure determination, data collection was performed on a 300 kV Titan Krios G4 equipped with a cold field emission gun and a post‐column Selectris energy filter (ThermoFisher) with a 5 eV slit width and a Falcon 4 or Falcon 4i direct electron detector (ThermoFisher). Images were collected at a pixel size of 0.745 Å/pix or 0.951 Å/pix with a cumulative dose of 40e⁻/Ų for untilted, and 50e−/Å2 for 30° tilted images, in eer format, using a defocus range in 0.3 μm increments (Appendix Fig S2).