Protein preparation steps for homology models

The N Protein N-terminal RNA binding domain model structure was built with the homology model module in YASARA (Krieger et al., 2009) using a series of templates from the PDB. These structures were obtained from a BLAST search of the N Protein sequence (UniProtKB accession #{"type":"entrez-protein","attrs":{"text":"P59595","term_id":"30173007","term_text":"P59595"}}P59595) (https://covid-19.uniprot.org/uniprotkb/{"type":"entrez-protein","attrs":{"text":"P59595","term_id":"30173007","term_text":"P59595"}}P59595#Names%20&%20Taxonomy) on the PDB sequence database. The model was built from selected template structures with sequence homology to N Protein: SARS-CoV-2 nucleocapsid protein N-terminal binding domain (PDB ID:6M3M) (Kang et al., 2020); RNA binding domain of nucleocapsid phosphoprotein from SARS-CoV-2 (PDB ID: 6VYO) (https://www.rcsb.org/structure/6VYO); C-terminal dimerization domain of Nucleocapsid Phosphoprotein from SARS-CoV-2 (PDBID: 6WJI) (https://www.rcsb.org/structure/6WJI); and the N-Terminal binding domain of the SARS-CoV-2 nucleocapsid phosphoprotein (PDBID: 6YI3) (Dinesh et al., 2020). Using these four structures as templates, a hybrid model for the N-terminal RNA binding domain of N protein was built in YASARA. Figure 1A shows the hybrid model generated for SARS-CoV-2 N Protein. In addition, a full-length model consisting of the entire N Protein structure was also built using the I-TASSER Server (Yang et al., 2015). Figure 1B shows the full-length model generated for SARS-CoV-2 N Protein. Both these N Protein models were further validated using structure evaluation servers, including ERRAT (Colovos & Yeates, 1993), VERIFY-3D (Eisenberg et al., 1997), the servers in the Structural Analysis and Verification Server (SAVES) (Lüthy et al., 1992), and QMean (Benkert et al., 2011) (Supplementary Figure S2). Then, the model structures were prepared as described above before running POOL (Tong et al., 2009; Somarowthu et al., 2011; Somarowthu & Ondrechen, 2012) and docking.

(A) Homology model built on YASARA for N-terminal domain of the Nucleocapsid protein shown in cyan. (B) Homology model built on I-TASSER for the full length Nucleocapsid protein with domains colored as: N-terminal domain—cyan; C-terminal domain—blue; linker region—lavender; other regions—gray.