Computational protocol: Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

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Protocol publication

[…] The initial apo hAgo2 conformation was taken from the crystal structure of hAgo2 in complex with miR-20a (PDB ID: 4F3T)[], and the missing residues were added using MODELLER[–]. The apo hAgo2 protein was solvated in a dodecahedron box containing 42,847 SPC water molecules[] with 129 Na+ ions and 159 Cl- ions to neutralize the charge. All the MD simulations were performed using the GROMACS 4.5.4 package[] and the Amber99SB-ILDN force field[]. Long-range electrostatic interactions were treated with the Particle-Mesh Ewald method[]. Both short-range electrostatic interactions and van der Waals interactions used a cutoff of 10Å. All bonds were constrained by the LINCS algorithm[]. Velocity-rescaling thermostat[] and the Parrinello-Rahman barostat [] were used for temperature and pressure coupling respectively. The system was first energy minimized with the steepest descent algorithm and equilibrated for 1ns under NPT ensemble (T = 310K, P = 1atm) with the positions of all the heavy atoms restrained. Next, we performed a 5-ns NPT simulation (T = 310K, P = 1atm) to further equilibrate the system. Finally, all the production MD simulations were performed under NVT ensemble at 310K.After the equilibration, we first performed six independent MD simulations ranging from 50 to 100ns which summed up to ~400ns. We then divided the conformations obtained from these six MD simulations (saved every 20ps with a total of ~20,000 conformations) into 20 clusters using the K-centers algorithm[]. We randomly selected one conformation from each cluster and initiated a second round of 150-ns MD simulations with one simulation from each of these 20 conformations. Next, we extracted ~170,000 conformations from the first two rounds of MD simulations and built an MSM containing 553 microstates that were further lumped into 9 macrostates. However, the 553-microstate MSM predicted kinetics inconsistent with the original MD simulations, indicating the necessity of additional sampling (see ). We thus seeded the third round of MD simulations from conformations taken from each of the 9 macrostate in the initial MSM with the number proportional to their predicted equilibrium populations. In this round, we performed 30 150-ns MD simulations with a saving interval of 20ps. Combining MD simulations from all three rounds, we obtained a dataset containing over 394,000 conformations extracted from ~8μs MD simulations. […]

Pipeline specifications

Software tools MODELLER, GROMACS, P-LINCS
Application Protein structure analysis
Organisms Homo sapiens
Diseases Genetic Diseases, Inborn