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the previous supposition ( Sections 2 and 3 ) that a Mg 2 þ ion, in a bridging
position, plays an active chemical role by interacting with the leaving group
(C1.1:O5 0 ) and general acid (G8:O2 0 ).
4.3. Simulation setup and protocols
The simulation setup and protocols are similar to those in Sections 2.3.1 and
2.3.2 , except the following modification and extension:
The all-atomCornell et al. force field (parm94) 143 in CHARMM format
is provided in the AMBER 9 package. 144-146 The equilibration procedures
have been extended to total 10 ns as follows: the positions of the solute
atoms, including the Mg 2 þ ion, were restrained by a harmonic potential
of 50 kcal/mole/ ˚ 2 in the equilibration stages.
4.3.1 Pre-annealing stage
Water and ion molecules were first energy-optimized for 2000 steps and
then underwent a constant volume simulation annealing: The temperature
was increased from 0 to 298 K at the rate of 1 K per ps. The system then was
kept at 298 K for 500 ps.
4.3.2 Solvent annealing stage
First step: The temperature increased from 298 to 600 K at the rate of 1 K/ps
and then was kept at 600 K for 500 ps with constant volume. Second step:
The temperature decreased from 600 to 298 K at the rate of 1 K/ps and then
was kept at 298 K for 1500 ps with constant volume. Third step: The system
was kept at 298 K for 3000 ps at a constant pressure (1 atm). The whole
annealing stage was repeated twice before the post-annealing stage.
4.3.3 Solute relaxation stage
After the annealing stage, the solute atoms were energy-optimized and then
were allowed to move under harmonic restraints over 500 ps simulation at
298 K with a 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 500 ps with a half-life decay parameter of 100 ps. At the
end of the 500 ps simulation, the restraints were reduced to about 3% of
the initial restraint values.
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