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In [16], variable stiffness springs are used, which allows adjustment of the point of equilibration.
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Moreover, with the new apparatus it is easy to determine the end point of the equilibration.
The end point of this equilibration run was then used as a starting point for subsequent EVB simulations.
The second stage of equilibration with variamine blue in presence of sodium acetate was found to be completed in about 10 min of equilibration.
However, considerable uncertainty still exists related particularly to the degree of equilibration of the pore water with the solids during preparation (pre-equilibration) and percolation of the column.
After equilibration in the isothermal-isobaric ensemble, an additional stage of equilibration was performed in the microcanonical ensemble.
We refer to the end point of this wild-type equilibration as the "equilibrated reference system", as it served as a common input structure to all of our computational experiments.
Thirty minutes of equilibration period were observed before each measure.
The starting point for our EVB simulations was the end point of the initial 10 ns equilibration run.
For each data point, Fe-cMyco or cMyco was added and after five minutes of equilibration at 25°C, the change in fluorescence was recorded using Perkin Elmer Fluorimeter LS55.
Each titration point was obtained by integration of the signal over 30 s after 1 1.5 minutes of equilibration.
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