(0) In this exercise you will harvest the experience you gained on
    natural degrees of freedom in WP2 and on optimization algorithms in WP3 to
    to optimize the conformation of fragments in 2cky (pdb code) riboswitch.
    Riboswitches are non coding segments (or untranslated regions
    UTRs) of an mRNA and they are responsible for directly binding to and
    sensing the presence of small metabolites resulting in change of gene
    expression encoded by the mRNA. More information can be found in the
    article that describes the structure determination of the 2cky riboswitch.

    S.Thore et al. (2006). Structure of the eukaryotic thiamine
    pyrophosphate riboswitch with its regulatory ligand.
    Science, 312, 1208-1211

    To start with, please open the figure (image file WP4/figure.jpg).
    Figure (a) shows the secondary structure of 2cky chain A. In this chain
    there are 5 helical (containing base pairs) (P1 - P5) and 5 loop (no base
    pairing of nucleotides) (L1 - L5) regions. Figure (b) describes
    the region definition for P1. A regional decomposition for the whole chain
    is also provided: WP4/examples/2cky_A/region.data. For this exercise
    we chose two chain fragments (2cky_j23 and 2cky_j124), whose secondary
    structure is shown in figure (c).

(1) As usual, please set up your links to "mosaics.x", pot_database,
    top_database libraries so that your WP4/examples library has this structure

    drwxr-xr-x  2cky_A
    drwxr-xr-x  2cky_j124
    drwxr-xr-x  2cky_j124_opt
    drwxr-xr-x  2cky_j23_opt_L1
    drwxr-xr-x  2cky_j23_opt_P2_P3
    lrwxr-xr-x  mosaics.x -> ../../MOSAICS/version.3.9.1_bgq/examples/mosaics.x
    lrwxr-xr-x  pot_database -> ../../TOPPOT/pot_database/
    lrwxr-xr-x  top_database -> ../../TOPPOT/top_database/

    where the paths to the executable may vary whether you have installed
    mosaics own your own or used an executable already available in your
    desktop.

(2) Based on techniques you learned in WP2 and WP3, please design a
    protocol to find the optimal (lowest energy) conformation of loop L1
    shown in figure (c).

    Please work in directory 2cky_j23_opt_L1, where we already set up
    an example optimization protocol for you to start with but we ask
    you to build the region file

     2cky_j23_opt_L1/region/region.opt_L1.data

    for this type of optimization. If you open this file you could see
    that we gave you the overall structure but we ask you fill in the
    missing lines.
    Hint: You could copy and paste quite a few lines from
          2cky_A/region.data.

    Once you have the file you can run a few optimization trajectories
    each can be represented by a plot of the potential energy vs the
    number of MC iterations.

    Please make one overall plot with the best (you found the lowest energy
    value) three optimization trajectories you obtained.

(3) Repeat the same exercise, but optimize the whole structure of 2cky_j23
    fragment and please work in directory 2cky_j23_opt_P2_P3. As a first
    step you have to complete the region file:

    2cky_j23_opt_P2_P3/region/region.opt_P2_P3.data

    As you could see in the structure we provide we recommend that you
    join regions P3 and L3.

    As before, please make a summary plot with your top three optimization
    trajectories (energy vs. number of steps).

(4) Run high temperature "entropic sampling" of the conformational space of
    fragment 2cky_j124. In this final exercise, we provide all the files you
    need including the initial setup in directory 2cky_j124.

    Visualize your trajectories using Pymol or VMD software packages.
    Plot the RMSD (Root-Mean-Square Deviation) of your visited conformational
    states from the initial structure. We recommend you to download the ANALYSE
    tools provided for this work package.

(5) Please switch to directory 2cky_j124_opt
    Run long optimization trajectories (same \simulation_typ and \minimize_type
    you used for (2) and (3)) for the 2cky_j124 fragment. Use stsamc period so
    that you have at least 5 full period temperature fluctuations. Depending on
    the speed of your desktop, perform N (5, 10, 20) x 100,0000 production steps.
    I would suggest running some preliminary shorter N x 10,000 trajectories to
    to find the best amplitude (\stsamc_ampl) so that you get trajectories
    visiting the lowest energy conformations. This may be a good set of initial
    parameters to start with

    \simulation_typ{MIN}
    \stsamc_type{trigonom}
    \stsamc_period{50000}
    \stsamc_ampl{1200}
    \stsamc_shift{0}
    \total_step_mc{250000}  2000 100            number > 1
    \statistics_freq{2000} 10

    Please analyse your production run and generate the following figures:
    (You may find the provided ANALYSE tools useful)

    (a) Plot the RMSD from initial structure
    (b) Plot the RMSD distribution
    (c) Plot the ENERGY vs RMSD
    (d) Overlay your figure (b) over your figure (c)