Analysis of the T-REMD trajectories in the context of the crystal structure of the VS ribozyme

To investigate the T-REMD trajectory at 300 K of a given KL complex in the context of the crystal structure of the VS ribozyme, composite models were built that merged the KL interaction of an individual T-REMD frame with a substrate/ribozyme complex, termed S_X/R_X, taken from the crystal structure of the VS ribozyme A₇₅₆G variant [PDB ID: 4R4P (27)], in which S_X is the SLI substrate of one protomer and R_X the helical domains II–VI of the other protomer. First, each frame of the T-REMD trajectories was aligned on the crystal structure by superposing the four core base pairs of stem V (residues 685–688 and 698–701 in the crystal structure; residues 5–8 and 18–21 in the I/V KL complex; residues 5–8 and 19–22 in the L88/L22 KL complex and residues 5–8 and 19–22 in the TAR/TAR* KL complex). Subsequently, the internal loop and stem Ia of SLI from the crystal structure was added to the model, superposing stem Ib from the crystal structure (residues 623–625 and 635–637) on the corresponding residues of the I/V, L88/L22 and TAR/TAR* KL complexes (residues 4–6 and 18–20, 4–6 and 17–19 and 4–6 and 17–19, respectively). From these superpositions, d_PP values were calculated by measuring the distance between phosphorus atoms of the scissile phosphate from the crystal structure and the composite model derived from a given frame of the T-REMD trajectory. Based on the same superpositions, heavy atom RMSD values were calculated between the G₆₃₈ loop and closing base pairs (residues 619–623 and 637–640) of the crystal structure and the frame-derived model.