A region surrounding the SARS-CoV2 PRF (Genomic coordinate: 12886-13635) was used to model the structural ensemble of the PRF. The region boundaries were determined based on base-paring probabilities output from partition function calculation performed in the SuperFold pipeline. Specifically, we ensured all nucleotides involved in base-pairing interactions with the PRF were included for the ensemble modeling.

To perform ensemble structure modeling, we followed step 6, 7 and 8 from the Rsample program (Spasic et al., 2018). To elaborate, first we used the Partition program (implemented in RNA structure v6.1, Mathews (Mathews, 2004)) to generate the partition saved file (PFS) for the region described. Replicate 1 SHAPE reactivity was used as a soft constraint (using the same slope and intercept as we used in the Superfold prediction) and the pseudoknotted base pairs were forced single strand. The PFS file was used to sample 1000 probable structures in proportion to their Boltzmann weights using the stochastic program (implemented in RNA structure v6.1) (Ding and Lawrence, 2003). This sample was then clustered using the hierarchical divisive method (Ding et al., 2005) and was asked to output 10 clusters with a representative conformation. A cluster is defined as a subset of structures with similar base pairs. The PFS file was visualized using IGV v2.8.2(Busan and Weeks, 2017).

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