SARS-CoV-2 PLproC111S crystals were flash-frozen in liquid nitrogen and then transferred into a dry nitrogen stream at 100 K for X-ray data collection. The X-ray data were collected on beamline BL17U1 at Shanghai Synchrotron Radiation Facility (SSRF) at 100 K and at a wavelength of 0.9792 Å using an Eiger X 16M image plate detector. Data were processed and scaled by using the program XDS (Kabsch, 2010). Experimental phase information was determined by the single anomalous dispersion (SAD) method using native Zinc anomalous signal. Phases and experimental electron density maps were calculated with the Phenix AutoSol (Terwilliger et al., 2009) within Phenix 1.18.2 (Liebschner et al., 2019). Iterative model building and refinement was completed with Coot 0.8.9 (Emsley et al., 2010) and Phenix REFINE (Afonine et al., 2012). The final Rwork and Rfree values were 18.47% and 21.34%. All of the residues are visible in electron density maps. The two monomers in the asymmetric unit superimpose with an rms deviation of 0.6 Å for all Cα atoms. The major difference between two monomers is the angle between the N-terminal Ubl domain and the catalytic which is mostly induced by crystal packing. The thumb and palm domains where the active site is located are almost identical.

PLproC111S-YM155 diffraction data were collected on beamline X06SA at Swiss Light Source (SLS) at 100 K and at a wavelength of 1.0000 Å using an Eiger X 16M image plate detector. The PLproC111S-YM155 structure was solved by molecular replacement (MR) with the PHASER (McCoy et al., 2007) within Phenix 1.18.2 (Liebschner et al., 2019) using the apo structure as a search template. The model from MR was subsequently subjected to iterative cycles of manual model adjustment with Coot 0.8 (Emsley et al., 2010) and refinement was completed with Phenix REFINE (Afonine et al., 2012). The final Rwork and Rfree values were 18.66% and 21.86%. All of the residues are visible in electron density maps. The structure reveals electron density for five YM155 molecules in one asymmetric unit. There are two consistent YM155 binding sites on each monomer: one at the substrate-binding pocket and one at the thumb domain. Another extra YM155 binding site was observed on the zinc-finger motif of molecule B. It is located at the interface between B chain and the neighboring A chain from another asymmetric unit, implying that this binding site might be induced by crystal packing.

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