Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 35

2.3.2.1.2 Annealing stage Four steps of constant volume simulations
(50 ps each) were performed in this stage. First step: The temperature in-
creased from 298 to 498 K in 7.5 ps and then was kept at 498 K. Second
step: The temperature increased from 498 to 698 K in 7.5 ps and then
was kept at 698 K. Third step: The temperature decreased from 698
to 498 K in 7.5 ps and then was kept at 498 K. Fourth step: The temper-
ature decreased from 498 to 298 K in 7.5 ps and then was kept at
298 K. The whole annealing stage was repeated three times before the
post-annealing stage.

2.3.2.1.3 Post-annealing stage Three steps of constant volume sim-
ulations were performed in this stage. First step (50 ps): The temperature
increased from 298 to 498 K in 7.5 ps and then was kept at 498 K. Second
step (50 ps): The temperature decreased from 498 to 298 K in 7.5 ps and then
was kept at 298 K. Third step (150 ps): The temperature was kept at 298 K
for 150 ps.

2.3.2.1.4 Solute relaxation stage The solute atoms were energy-
optimized and then were allowed to move under harmonic restraints over
a 50 ps simulation at 298 K under constant pressure of 1 atm. The harmonic
force constant (in kcal/mol/ ˚ 2 ) on each heavy atom was obtained from the
empirical formula k i

10 3 / B i where k i is the force constant for atom i
and B i is the corresponding crystallographic B -value. The restraints were
exponentially released over 50 ps with a half-life decay parameter of 10 ps.
At the end of the 50 ps simulation, the restraints were reduced to about 3%
of the initial restraint values. Three harmonic restraints of 20 kcal/mol/ ˚ 2
were added to keep the Mg 2 þ ion in the middle of the C1.1:O2P and A9:
O2P positions. Another harmonic restraint of 20 kcal/mol/ ˚ 2 was used to
force the distance between G8:H O2P and C1.1:O5 0 to be around 1.8 ˚ ,
which is to ensure that the HO2P of G8 is initially hydrogen bonded. All
restraints were then released prior to the production simulation.

¼ 25 þ 2

2.3.2.2 Production simulation
After the 1 ns of solvent equilibration, the whole system was energy-
optimized and unconstrained dynamics simulation began from 0 K under
constant pressure of 1 atm. The temperature was increased to 298 K at
the rate of 1 K/ps and then kept fixed at 298 K. The same equilibration pro-
cess was applied for each simulation. A total of 12 ns of unconstrained
dynamics was performed for each of the eight simulations (reactant with
and without Mg 2 þ , ETS mimic, and LTS mimic), the last 10 ns of which