High-Resolution Cryo-EM of SARS-CoV-2 RdRp

Data collection and preprocessing was the same as previously described^{11 (link)}. Briefly, data was collected using SerialEM^{37 (link)} on a 300 keV Titan Krios transmission electron microscope (Thermo Fischer Scientific) and a K3 direct electron detector (Gatan). Inelastically scattered electrons were filtered out prior to detection using a GIF quantum energy filter (Gatan) using a slit width of 20 eV. Images were acquired at a nominal magnification of 105,000x and a calibrated pixel size of 0.834 Å/pixel. Due to previously observed preferred orientation when imaging RdRp complexes^{10 (link)}, data was collected using a 30° tilt to obtain more particle orientations. 7043 raw micrographs were acquired in total and preprocessed on-the-fly in Warp for automatic contrast transfer function (CTF) estimation, averaging, motion correction, and particle prediction and extraction. 2.2 million individual RdRp particles were predicted and exported by Warp and imported into cryoSPARC and subjected to a Hetero Refinement job using five ‘Junk‘ classes and one class representing monomeric RdRp as described previously^{11 (link)}. The resulting particle set was used for a 3D homogeneous refinement to obtain refined poses and positions for each RdRp monomer.