Fei tecnai f20 electron

A total of 257 micrographs from a cryo-grid of ΦKZ nvRNAP were collected on an early Falcon direct electron detector using an FEI Tecnai F20 electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) at a magnification of 81 000-fold, an acceleration voltage of 200 kV, and a total dose of 50 e^–/Ų over a defocus range of –2 to –5 μm. A dataset of 45 980 particles was selected semi-automatically using BOXER (22 (link)). The parameters of the contrast transfer function were determined using CTFFIND4 (23 (link)). Particles were 2D-classified into 100 classes in two dimensions using RELION 3.0 (24 (link)) and seven well-defined classes including 18 508 particles were selected for initial 3D reconstruction. Initial models were generated using the stochastic gradient descent approach in RELION 3.0, then filtered to 50 Å and used as an initial reference for automatic refinement. Projections from the resulting initial models were consistent with class-averages, and were therefore used for further refinement of higher resolution data.

HCoV-229E was produced by inoculating a confluent Huh-7 T75 flask at an MOI of 0.008 in DMEM supplemented with 5% FBS and kept at 33°C and 5% CO₂ for 5 days. Supernatant was harvested and treated with DMSO or 10 μM Pba and further kept in the dark or exposed to light for 30 min. NaCl then was added to a final concentration of 400 mM to induce an osmotic shock. Viruses were fixed in 4% PFA. For cryo-EM experiments of the particles, lacey carbon Formvar 300 mesh copper grids were used after a standard glow-discharged procedure. Plunge freezing was realized using the EM-GP apparatus (Leica). Specimens were observed at −175°C using a cryo holder (626; Gatan) with a ThermoFisher FEI Tecnai F20 electron microscope operating at 200 kV under low-dose conditions. Images were acquired with an Eagle 4k by 4k camera (ThermoFisher FEI).