GIS mapping
In the present study, Qgis V 2.18.26 software was used for the mapping and visualization of the reported cases of SARS-COV-2 infection reported globally till 16.4.2020 retrieved from NCBI database. We did not use the exact co-ordinate for framing as we do not have the exact GPS coordinates of the cases, who have been participated for the whole genome sequencing. All the cases are tagged with a different legend to show the vast spreading of the Covid-19 outbreak and the NCBI accession numbers are assigned to each legend (Fig. 1).
Phylogenetic analysis of spike protein of SARS-CoV- 2
To study the evolutionary pathway of the SARS-CoV-2, three phylogenetic trees were constructed. In the first Phylogenetic analysis, the spike protein (Surface glycoprotein) amino acid sequences of various SARS virus were retrieved from NCBI-Genbank database (Table 1). The sequences were aligned using Clustal W alignment program and the tree was constructed using Neighbor joining alignment. Bootstrapping was performed at 1000 replications of MEGA 7 program (Kumar et al. 2016). Tilapia lake virus hypothetical protein having genebank accession number MN094791 was kept as an out group
The second Phylogenetic tree was constructed to determine the evolutionary relationship of mostly reported human Coronavirus viz. MERS, SARS-CoV and SARS-CoV-2. The S protein amino acid sequences were retrieved from NCBI database and were aligned. The evolutionary tree was generated using Neighbor joining program of MEGA 7 (Kumar et al. 2016) and bootstrapping was performed at 1000 replications. Tilapia lake virus hypothetical protein was kept as an out group.
Since SARS-CoV2 infection have been reported to be pandemic by WHO (https://www.who.int/emergencies/diseases/novel-coronavirus-2019) so the third Phylogenetic tree was constructed to check whether there is any variation between the S protein that have been reported globally. For the construction of the evolutionary tree, almost every representative sequence of Spike protein reported till 16.4.2020 from various geographical locations including Asia, Africa, North America, South America and Europe were retrieved from the new NCBI virus database (Table. 2) providing inclusive information regarding SARS-CoV2 (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?VirusLineage_ss=Severe%20acute%20respiratory%20syndrome%20coronavirus%202,%20taxid:2697049&SeqType_s=Nucleotide). The sequences were aligned using Neighbor joining program and bootstrapping was performed at 1000 replications of MEGA 7 program (Kumar et al. 2016). Tilapia lake virus hypothetical protein was kept as an out group.
Molecular Docking between SARS-Cov-2 spike glycoprotein and ACE2 receptor
The protein structure of SARS-Cov-2 spike glycoprotein having PDB ID-6VXX was retrieved from PDB database. Molecular docking between the SARS-CoV-2 spike glycoprotein (PDB ID: 6VXX) protein and ACE2 (PBD ID: 108A) protein are furthermost vital process to characterize the molecular interaction and accurate bonding pattern of the proteins. This is an essential process for supporting the binding affinity towards the outer spike glycoprotein and human ACE2 receptor. Molecular docking determines the cellular functions of the proteins (Lavi et al. 2013). In the present study, PatchDock docking server was used for carrying out molecular docking analysis (Duhovny et al. 2002). The server is having a geometric complimentary based algorithm to achieve the exact molecular docking (Yadav et al. 2017). From this server we also evaluated interface area, geometric score and Atomic Contact Energy (ACE) of the docking complexes along with the generated PDB file for analysis the proper molecular interactions. The RasMol software v.2.7.4.2 was helped to visualize the generated PDB file of protein-peptide docking complex (Chen et al. 2009).
Molecular Dynamic Simulation
Normal mode analysis (NMA) is a powerful method for predicting the possible large-scale movements of a specified bio-macromolecule (Prabhakar et al. 2016).This specific analysis has used in broad field of structural biology, like the study of conformational changes of protein binding to ligand, changing of conformation opening and closure structural stability of membrane channel protein, potential actions of the ribosome, and also calculate viral capsid maturation. The iMODS server was employed for representation of the dynamic motion of the docking complex accordingly with the help of NMA (López-Blanco et al., 2014). This server measures the different parameters regarding molecular structural dynamics like; deformability plot, B-factor, eigen value, Covariance matrix. Result of deformability plot reveals the specific region of protein (coiled regions) while the B-factor shows atomic deformation. Eigen value calculates rigidity of molecular motion. Co-variance matrix signifies the correlated atomic pairs depending on specific color code.
Homology modeling and molecular docking of Spike protein
In order to generate the 3D structure of spike protein, homology modeling was carried out at Swiss Model server (Waterhouse et al. 2018). Amino acid sequence having accession number viz. MT049951; MT12098, MT007544, NC045512, MT019532 were retrieved from NCBI Genbank server. Spike protein structure having PDB ID- 6VXX was used as template for the generation of the model. Validation of the generated model was carried through Q- mean score (Benkert et al. 2011) and Ramachandran plot analysis.
To characterize the molecular interaction and accurate bonding pattern of the designed proteins, the generated 3D protein structure were docked with human ACE2 (PBD ID: 108A) receptor protein. PatchDock docking server was used for carrying out molecular docking analysis (Duhovny et al. 2002). The server can also evaluate interface area, geometric score and Atomic Contact Energy (ACE) of the docking complexes.