Exploring hRPA Dynamics and Flexibility

To probe the large-scale functional motions of the modeled hRPA and its dynamics and flexibility, we performed the Normal Mode Analysis (NMA). For NMA analysis, the initial structure of the protein was selected after discarding the initial few steps at ϵ_inter value of 0.3 kcal/mol and 1.0 kcal/mol. Here, we used the coarse-grained structure of ssDNA binding core domains only (domains A-E). To perform the NMA analysis and produce the visualizations of the data, the Bio3D package in R was used [74 (link)]. A variety of functions from the Bio3D package were employed. Some of them are nma.pdbs(), dccm.nma(), and pymol(). The function nma.pdbs() was used to calculate the normal modes and produce an NMA object for visualization. dccm.nma() function was used to calculate the cross-correlation between DBDs. This function utilizes the normal mode analysis of a protein structure to calculate a cross-correlation matrix with elements C_ij. Graphically, it is represented by dynamic cross-correlation map (DCCM). The C_ij value corresponds to the fluctuations of residues i and j and denotes if they are correlated, anti-correlated or uncorrelated. For example, C_ij = 1 signifies their fluctuations are completely correlated, whereas, a completely anti-correlated fluctuations of two interacting beads is represented by C_ij = -1. C_ij = 0 indicates that the fluctuations of two beads are not correlated. The Bio3D package also allows the visualization of the normal modes either through a vector field representation or trajectory file in PyMol using the Bio3D inbuilt function, pymol(). With this function, three-dimensional visualization of the protein is obtained and it highlights the domain dynamics in the hRPA molecule. The total number of modes generated were 2055 including the first 6 modes with zero frequency (trivial modes). These trivial modes correspond to the rotational and translational modes. For comparison between the normal modes of the protein in apo hRPA and bound hRPA conformations obtained from MD simulation trajectory (where binding is complete), we calculated the root-mean-square inner product (RMSIP) of the normal modes obtained from normal mode analysis of both conformations. These calculations were performed to facilitate a direct comparison of the flexibility patterns between protein structures. To determine which normal modes contribute to the observed conformational changes in hRPA protein from our MD simulation trajectory, the squared overlap analysis was carried out. Here, the squared overlap of the first 20 modes was calculated, with the conformational difference vector between the first (after discarding the initial 10⁴ steps) and last frames (the bound hRPA-ssDNA conformation) of our MD trajectories. The trajectory of MD simulation consists of the unbiased binding simulation of hRPA and ssDNA to achieve the final bound complex of ssDNA and hRPA (bound state) starting from both molecules in solution (unbound state). All these calculations were performed using the Bio3D package in R [74 (link)–76 (link)].