Qualitative Estimation of Protein–Ligand Complex Stability through Thermal Titration Molecular Dynamics Simulations

The prediction of ligand efficacy has long been linked to thermodynamic properties such as the equilibrium dissociation constant, which considers both the association and the dissociation rates of a defined protein–ligand complex. In the last 15 years, there has been a paradigm shift, with an increased interest in the determination of kinetic properties such as the drug–target residence time since they better correlate with ligand efficacy compared to other parameters. In this article, we present thermal titration molecular dynamics (TTMD), an alternative computational method that combines a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein–ligand interaction fingerprints for the qualitative estimation of protein–ligand-binding stability. The protocol has been applied to four different pharmaceutically relevant test cases, including protein kinase CK1δ, protein kinase CK2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease, on a variety of ligands with different sizes, structures, and experimentally determined affinity values. In all four cases, TTMD was successfully able to distinguish between high-affinity compounds (low nanomolar range) and low-affinity ones (micromolar), proving to be a useful screening tool for the prioritization of compounds in a drug discovery campaign.

.Principal chemical properties of the CK1 ligands utilized in this work. For each ligand, the molecular weight, the number of hydrogen bond acceptors and donors, and the number of rotatable bonds are reported. For reference, the average MS coefficient extracted from the pool of TTMD simulations is also reported.     Figure S2: Analyses performed on a representative TTMD trajectory (MD5) for the complex Data Bank with accession code 4TN6. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD sim is reported on the horizontal axis, while the IFP on the vertical axis.

Structure
: Analyses performed on a representative TTMD trajectory (MD5) for the complex Data Bank with accession code 4TN6. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP mensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 4TN6. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value mensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the , while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand ulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S3: Analyses performed on a representative TTMD traje Data Bank with accession code 5IH5. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the strai being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagr is reported on the horizontal axis, while the IFP on the vertical axis. : Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 5IH5. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the strai -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the eported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported ctory (MD2) for the complex deposited in the Protein Data Bank with accession code 5IH5. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the eported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand een) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S4: Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 5IH6. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagre (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongsid TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP on the vertical axis.
a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 5IH6. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongsid TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 5IH6. A) superposition between the ligand conformation sampled in the last trajectory en). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S5: Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 5MQV. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while t upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 5MQV. A) superposition between the ligand conformation sampled in the last trajectory ligand binding mode (seagreen). B) titration profile: the average IFP units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 5MQV. A) superposition between the ligand conformation sampled in the last trajectory ligand binding mode (seagreen). B) titration profile: the average IFP CS value units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the he simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S6: Analyses performed on a representative TTMD trajectory (MD3 Data Bank with accession code 2ZJW (neutral form). A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration prof value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD3) for the complex deposited in the Protein Data Bank with accession code 2ZJW (neutral form). A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration prof value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported ) for the complex deposited in the Protein Data Bank with accession code 2ZJW (neutral form). A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S7: Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 2ZJW (monocharged). A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand b IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of th straight being reported in the legend. C) time energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacte the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP reported on the vertical axis.
d on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 2ZJW (monocharged). A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) time-dependent per-residue decomposition of the protein energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacte the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the igand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are d on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 2ZJW (monocharged). A) superposition between the ligand conformation sampled in inding mode (seagreen). B) titration profile: the average value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form e TTMD simulation, with the slope of the residue decomposition of the protein-ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. dependent evolution of the RMSD for both the igand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation (adimensional units) and RMSD (Å) value, respectively, are Figure S8: Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 3H30. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the star being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contrib TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 3H30. A) superposition between the ligand conformation sampled in the last trajectory and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, : Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 3H30. A) superposition between the ligand conformation sampled in the last trajectory and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue t and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, ution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S9: Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 3PE1. A) superposition between the ligand conformation sampled in the frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 3PE1. A) superposition between the ligand conformation sampled in the frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue traight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteinaals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 3PE1. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue traight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, aals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) RMSD (Å) value, respectively, are reported Figure S10: Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 3PE2. A) superposition between the ligand frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in th dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IF on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 3PE2. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in th dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) lution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, ed as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 6HOU. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) orizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein position between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value e step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein accession code 4MP2. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein accession code 4MP2. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the he simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S13: Analyses performed on a representative TTMD trajectory (MD Data Bank with accession code 4V25. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the avera (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) thro is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 4V25. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the avera (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the n the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported 4) for the complex deposited in the Protein Data Bank with accession code 4V25. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the n the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand ughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S14: Analyses performed on a repres Data Bank with accession code 5J71. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD1) for the complex deposited in the Protein Data Bank with accession code 5J71. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the MD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand ein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported entative TTMD trajectory (MD1) for the complex deposited in the Protein Data Bank with accession code 5J71. A) superposition between the ligand conformation sampled in the last trajectory titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the MD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand ein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported alyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 5M4M. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contact TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported alyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 5M4M. A) superposition between the ligand conformation sampled in the last trajectory binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue he TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD3) for the complex deposited in the Protein Data Bank with accession code 7EA0. A) superposition between the ligand conformation sampled in the last trajectory (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respe : Analyses performed on a representative TTMD trajectory (MD3) for the complex deposited in the Protein Data Bank with accession code 7EA0. A) superposition between the ligand conformation sampled in the last trajectory (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) volution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S17: Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 6M2N. A) superposition between the ligand conformation sampled i frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 6M2N. A) superposition between the ligand conformation sampled i frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteinder Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 6M2N. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) s) and RMSD (Å) value, respectively, are reported Figure S18: Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 7LTJ. A) superposition between the l frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein Data Bank with accession code 7LTJ. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) nt evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD5) for the complex deposited in the Protein igand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein Data Bank with accession code 7M8P. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported : Analyses performed on a representative TTMD trajectory (MD2) for the complex deposited in the Protein superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the he simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S20: Analyses performed on a representative TTMD trajectory (MD Data Bank with accession code 7M91. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the avera (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) thro is reported on the horizontal axis, while the IFP on the vertical axis.
: Analyses performed on a representative TTMD trajectory (MD1) for the complex deposited in the Protein Data Bank with accession code 7M91. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the avera (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the n the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported 1) for the complex deposited in the Protein Data Bank with accession code 7M91. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the n the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand ughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Figure S21: Analyses performed on a repres Data Bank with accession code 7N44. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the being reported in the legend. C) timedefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the TTMD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) upper: time-dependent evolution of the IFP (tomato) and the protein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP on the vertical axis.
Video V1: Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein target CK1δ (left: complex 3UZP TTMD replicate 3; video, the top portion reports the visual representation of the trajectory: the protein is depicted as tomato ribbons, while the ligand and protein residues within 5 state of the simulation is also reported, u instead, reports two different plots: the top one, reports the time and temperature dependent evolution of the IFP score, while the bottom one reports the time dependent evolut (tomato) RMSD.
Video V2: Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein target CK2 (left: complex 3PE2 TTMD replicate 5; video, the top portion reports the visual representation of the trajectory: the protein is depicted as tomato ribbons, while the ligand and protein residues within 5 state of the simulation is also reported, using the same representation style and seagreen as color. The bottom portion, instead, reports two different plots: the top one, reports the time and temperature dependent evol : Analyses performed on a representative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 7N44. A) superposition between the ligand conformation sampled in the last trajectory frame (tomato) and the reference ligand binding mode (seagreen). B) titration profile: the average IFP (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the -dependent per-residue decomposition of the proteindefined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the MD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the IFP CS score; lower: time-dependent evolution of the RMSD for both the ligand ein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) is reported on the horizontal axis, while the IFP CS (adimensional units) and RMSD (Å) value, respectively, are reported Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein : complex 3UZP TTMD replicate 3; right: complex 5IH6 TTMD replicate 4). Within each half of the isual representation of the trajectory: the protein is depicted as tomato ribbons, while the ligand and protein residues within 5 Å of the ligand are depicted as tomato licorice. For visual reference, the initial state of the simulation is also reported, using the same representation style and seagreen as color. The bottom portion, instead, reports two different plots: the top one, reports the time and temperature dependent evolution of the IFP score, while the bottom one reports the time dependent evolution of both the protein backbone (seagreen) and ligand : Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein : complex 3PE2 TTMD replicate 5; right: complex 6HOU TTMD replicate 2). Within each half of the video, the top portion reports the visual representation of the trajectory: the protein is depicted as tomato ribbons, while the ligand and protein residues within 5 Å of the ligand are depicted as tomato licorice. For visual reference, the initial state of the simulation is also reported, using the same representation style and seagreen as color. The bottom portion, instead, reports two different plots: the top one, reports the time and temperature dependent evol entative TTMD trajectory (MD4) for the complex deposited in the Protein Data Bank with accession code 7N44. A) superposition between the ligand conformation sampled in the last trajectory titration profile: the average IFP CS value (adimensional units) for each "TTMD step" is reported as a function of the step temperature (K) in the form of blue dots. A red straight line connects the start and the final point of the TTMD simulation, with the slope of the straight -ligand interaction energy, defined as a sum of the van der Waals and electrostatic contribution. The 25 most contacted residues alongside the MD trajectory are reported on the vertical axis, while the simulation time (ns) is reported on the horizontal axis. D) dependent evolution of the RMSD for both the ligand ein backbone (seagreen) throughout the TTMD simulation. In both plots, the simulation time (ns) (adimensional units) and RMSD (Å) value, respectively, are reported Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein : complex 5IH6 TTMD replicate 4). Within each half of the isual representation of the trajectory: the protein is depicted as tomato ribbons, while of the ligand are depicted as tomato licorice. For visual reference, the initial sing the same representation style and seagreen as color. The bottom portion, instead, reports two different plots: the top one, reports the time and temperature dependent evolution of the IFP CS ion of both the protein backbone (seagreen) and ligand : Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein TTMD replicate 2). Within each half of the video, the top portion reports the visual representation of the trajectory: the protein is depicted as tomato ribbons, while e. For visual reference, the initial state of the simulation is also reported, using the same representation style and seagreen as color. The bottom portion, instead, reports two different plots: the top one, reports the time and temperature dependent evolution of the IFP CS score, while the bottom one reports the time dependent evolution of both the protein backbone (seagreen) and ligand (tomato) RMSD.
Video V3: Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein target PDK2 (left: complex 4V25 TTMD replicate 4; right: complex 4MP2 TTMD replicate 4). Within each half of the video, the top portion reports the visual representation of the trajectory: the protein is depicted as tomato ribbons, while the ligand and protein residues within 5 Å of the ligand are depicted as tomato licorice. For visual reference, the initial state of the simulation is also reported, using the same representation style and seagreen as color. The bottom portion, instead, reports two different plots: the top one, reports the time and temperature dependent evolution of the IFP CS score, while the bottom one reports the time dependent evolution of both the protein backbone (seagreen) and ligand (tomato) RMSD.
Video V4: Comparison between the representative TTMD replicate for a weak and a strong binder in the case of protein target SARS-CoV-2 M pro (left: complex 7LTJ TTMD replicate 4; right: complex 7M91 TTMD replicate 4). Within each half of the video, the top portion reports the visual representation of the trajectory: the protein is depicted as tomato ribbons, while the ligand and protein residues within 5 Å of the ligand are depicted as tomato licorice. For visual reference, the initial state of the simulation is also reported, using the same representation style and seagreen as color. The bottom portion, instead, reports two different plots: the top one, reports the time and temperature dependent evolution of the IFP CS score, while the bottom one reports the time dependent evolution of both the protein backbone (seagreen) and ligand (tomato) RMSD.