Biology Reference
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2.3.1.3 System preparation
Initial structures used in the simulations were based on the crystallographic
structure of the first eHHR structure. 73 The positions of hydrogen atoms were
determined using the HBUILD facility in the programCHARMM 106 (version
c32a2). With hydrogen atoms built and the Mg 2 þ cation position established,
the ribozyme was immersed in a rhombic dodecahedral cell of 10,062 pre-
equilibrated TIP3P water molecules centered about the active site, and pruned
such that any water molecule within 2.8 ˚ from the solute was removed. The
ion atmosphere consisted of Na þ and Cl ions that were added at random posi-
tions to neutralize the system and reach the physiologic extracellular concentra-
tion of 0.14 M. The ion positions were kept initially at least 4.7 ˚ away from
any solute atoms. The resulting system (the reactant state) contained 9053 water
molecules, 82 Na þ and 23 Cl ions, and 2021 RNA atoms.
2.3.2 Simulation protocols
Periodic boundary conditions were used along with the isothermal-isobaric
ensemble (NPT) at 1 atm and 298 K using extended system pressure algo-
rithm 108 with effective mass of 500.0 amu and Nos´-Hoover thermo-
stat 109,110 with effective mass of 1000.0 kcal/mol-ps 2 , respectively. The
smooth particle mesh Ewald method 111,112 was employed with a
value
of 0.35 ˚ 1 , 80 FFT grid points for each of the lattice directions, and a
B-spline interpolation order of six. Nonbonded interactions were treated
using an atom-based cutoff of 10 ˚ with shifted van der Waals potential.
Numerical integration was performed using the leap-frog Verlet algorithm
with 1 fs time step. 113 Covalent bond lengths involving hydrogen were con-
strained using the SHAKE algorithm. 114
l
2.3.2.1 Solvent/ion equilibration
The following equilibration procedures (total 1 ns) were applied to the sys-
tem prior to the production simulations in order to insure reasonable relax-
ation of the solvent and ion environment. The positions of the solute atoms,
including the Mg 2 þ ion, were held fixed in the equilibration stages.
2.3.2.1.1 Pre-annealing stage Water and ion molecules were first
energy-optimized and then underwent a constant volume simulation
annealing for 50 ps. The temperature was increased from 0 to 298 K in a
7.5 ps period and then was kept at 298 K. The annealing simulations were
repeated twice with temperature increased from 298 to 498 K and then back
to 298 K.
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