<br><br><div class="gmail_quote">On Mon, May 10, 2010 at 6:29 PM, Justin A. Lemkul <span dir="ltr">&lt;<a href="mailto:jalemkul@vt.edu">jalemkul@vt.edu</a>&gt;</span> wrote:<br><blockquote class="gmail_quote" style="border-left: 1px solid rgb(204, 204, 204); margin: 0pt 0pt 0pt 0.8ex; padding-left: 1ex;">
<div class="im">Ok . Now I understood. I have one more doubt , as I mentioned to you, I am using one residue in the extracellular loop as a reference point. Since this is in the loop, do you think it can be good reference point (due to th large fluctuatuion in the loop, will it affect the result)? <br>

<br>
Aswathy wrote:<br>
<blockquote class="gmail_quote" style="border-left: 1px solid rgb(204, 204, 204); margin: 0pt 0pt 0pt 0.8ex; padding-left: 1ex;">
I am pulling through the channel with respect to a single residue on one &quot;side&quot;(extracellular) of the structure. I have used pull_geometry = distance &amp;  pull_dim =  N N Y. From this what I understood is ligand will pull along the z direction with respect to the reference group (away from r_57).  (i.e from extracellular to intracellular). Is this correct?<br>

<br>
</blockquote>
<br></div>
I don&#39;t think so.  If you are pulling through a channel, using an extracellular residue as a reference, you will be changing the sign of the distance, rendering &quot;pull_geometry = distance&quot; useless.  For example, in order to properly calculate the PMF, you have to pull from the aqueous solvent, into the channel, then back out into the solvent.  At some point, your ligand is outside the channel (such that, for example, the z-coordinate of the ligand is greater than that of r_57, so distance &gt; 0).  Then, as your ligand enters the channel, its z-coordinate is less than that of r_57, so distance &lt; 0.  If this is the case, you must use &quot;pull_geometry = position&quot; to get the correct signs, otherwise your umbrella sampling window reference distances will be nonsensical.<br>

<br>
-Justin<br>
<br>
<blockquote class="gmail_quote" style="border-left: 1px solid rgb(204, 204, 204); margin: 0pt 0pt 0pt 0.8ex; padding-left: 1ex;"><div class="im">
Here is my umbrella sampling .mdp parameters<br>
<br>
pull                     = umbrella<br>
pull_geometry            = distance<br>
pull_dim                 =  N N Y<br>
pull_start               = yes<br>
pull_nstxout             =  10<br>
pull_nstfout             =  10<br>
pull_ngroups             =  1<br>
pull_group0              =  r_57<br>
pull_group1              =  r_C1<br>
pull_k1                  =  1000<br>
pull_init1               =  0<br>
<br></div><div class="im">
On Mon, May 10, 2010 at 4:50 PM, Justin A. Lemkul &lt;<a href="mailto:jalemkul@vt.edu" target="_blank">jalemkul@vt.edu</a> &lt;mailto:<a href="mailto:jalemkul@vt.edu" target="_blank">jalemkul@vt.edu</a>&gt;&gt; wrote:<br>

<br>
<br>
<br>
    Aswathy wrote:<br>
<br>
        Thanks for your  reply.<br>
<br>
         In this case reference (r57) is not the part of the channel.<br>
        But it is a residue in the loop above the channel entry. Thats<br>
        why I used pull_geometry=distance. Therefore I am pulling the<br>
        ligand away from this reference.<br>
<br>
<br>
    So you are not pulling through the channel?  Or you are pulling<br>
    through the channel with respect to a single residue on one &quot;side&quot;<br>
    of the structure?  If your ligand ever crosses over this reference<br>
    in any way, the reference distance will change sign and thus Tom is<br>
    right, you should use &quot;pull_geometry = position.&quot;  With &quot;distance,&quot;<br>
    you can only ever have positive reference distances.<br>
<br>
    What are your .mdp settings during umbrella sampling?<br>
<br>
    -Justin<br>
<br>
        Thanks<br>
        -Aswathy<br>
<br>
<br>
        On Mon, May 10, 2010 at 3:05 PM, Thomas Piggot<br>
        &lt;<a href="mailto:t.piggot@soton.ac.uk" target="_blank">t.piggot@soton.ac.uk</a> &lt;mailto:<a href="mailto:t.piggot@soton.ac.uk" target="_blank">t.piggot@soton.ac.uk</a>&gt;<br></div><div><div></div><div class="h5">

        &lt;mailto:<a href="mailto:t.piggot@soton.ac.uk" target="_blank">t.piggot@soton.ac.uk</a> &lt;mailto:<a href="mailto:t.piggot@soton.ac.uk" target="_blank">t.piggot@soton.ac.uk</a>&gt;&gt;&gt; wrote:<br>
<br>
           Hi,<br>
<br>
           If you defined the reference (r_57) as part of your channel then<br>
           with pull_geometry=distance you will have problems as the<br>
        distance<br>
           between pull_group1 and pull_group0 becomes closer to zero<br>
        and then<br>
           the distance becomes positive again.<br>
<br>
           I recently had this with my umbrella sampling simulations. Search<br>
           for the discussion of things you can do to address this issue<br>
        on the<br>
           list. To stop this being a problem in the first place you should<br>
           have used pull_geometry=position.<br>
<br>
           Cheers<br>
<br>
           Tom<br>
<br>
           Aswathy wrote:<br>
<br>
               Can any one help me please? I looking forward to hear<br>
        from any<br>
               of you.<br>
               Thank you.<br>
<br>
<br>
               On Thu, May 6, 2010 at 1:19 PM, Aswathy<br>
        &lt;<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a> &lt;mailto:<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a>&gt;<br>
               &lt;mailto:<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a> &lt;mailto:<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a>&gt;&gt;<br>
        &lt;mailto:<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a> &lt;mailto:<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a>&gt;<br>
<br>
               &lt;mailto:<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a><br>
        &lt;mailto:<a href="mailto:ammasachu@gmail.com" target="_blank">ammasachu@gmail.com</a>&gt;&gt;&gt;&gt; wrote:<br>
<br>
                  Ok i will explain you in detail.<br>
<br>
                   Initially i pulled the ligand through the protein<br>
        channel ,<br>
               using<br>
                  the given parameters.<br>
<br>
                  pull                     = umbrella<br>
                  pull_geometry            = distance<br>
                  pull_dim                 =  N N Y<br>
                  pull_start               = yes<br>
                  pull_nstxout             =  10<br>
                  pull_nstfout             =  10<br>
                  pull_ngroups             =  1<br>
                  pull_group0              =  r_57<br>
                  pull_group1              =  r_C1<br>
                  pull_rate1               =  0.01<br>
                  pull_k1                  =  1500<br>
<br>
                  Then I extracted the frames from the trajectory using<br>
        the perl<br>
                  program provided with tutorial. COM distance I took as<br>
        nearly<br>
               0.12<br>
                  nm. (But sometimes I failed to obtain frames exactly<br>
        at that<br>
                  interval, but took  nearly at 0.12). Each frame I used for<br>
               Umbrella<br>
                  sampling for 1ns.<br>
                  Then I checked histograms for overlapping (Some<br>
        histograms were<br>
                  entirely overlapped and I removed that from the list,<br>
        where ever<br>
                  gaps i selected new frames and did sampling so that I<br>
        can get an<br>
                  evenly distributed histograms , I know this will<br>
        change the<br>
               overall<br>
                  COM distribution but is there any other way to solve<br>
        this?) .<br>
<br>
                  Finally once I obtained reasonably good overlapped<br>
        histograms, I<br>
                  plotted PMF using g_wham. The plot  was a steeply<br>
        increasing<br>
                  potential.  How can we get increased PMF even when the<br>
        ligand is<br>
                  reached out of the channel?<br>
<br>
<br>
                           Did I made any mistake any where , I am confused.<br>
<br>
                  Thank you.<br>
                  -Aswathy<br>
<br>
<br>
<br>
                  On Thu, May 6, 2010 at 12:56 PM, Jochen Hub<br>
               &lt;<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a> &lt;mailto:<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a>&gt;<br>
        &lt;mailto:<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a> &lt;mailto:<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a>&gt;&gt;<br>
                  &lt;mailto:<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a><br>
        &lt;mailto:<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a>&gt;<br>
               &lt;mailto:<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a><br>
        &lt;mailto:<a href="mailto:jochen@xray.bmc.uu.se" target="_blank">jochen@xray.bmc.uu.se</a>&gt;&gt;&gt;&gt; wrote:<br>
<br>
                      Aswathy wrote:<br>
<br>
<br>
                          Hi gromacs users,<br>
<br>
                          I am using Gromacs 4.0.4 package. I am doing<br>
        SMD of a<br>
               ligand<br>
                          transport through a channel.<br>
<br>
                          I performed SMD and did umbrella sampling<br>
        (Thanks to<br>
               Justin<br>
                          for his  tutorial). Extracted frames with a window<br>
               spacing<br>
                          interval  of ~0.12nm. and did 1ns sampling.<br>
               Histograms are<br>
                          with reasonabvle overlap. Then I used g_wham<br>
        for plotting<br>
                          PMF considering first 300ps as equilibration.<br>
<br>
                      Isn&#39;t SMD usually referred to pulling at some<br>
        finite pulling<br>
                      speed? That would not be umbrella sampling.<br>
<br>
                      Anyway, you&#39;ll have to provide a lot more data to<br>
        enable<br>
               us to<br>
                      help you.<br>
<br>
                      Jochen<br>
<br>
<br>
<br>
<br>
                          I am getting a plot , but potential is increasing<br>
                          constantly. ie, PMF is not converged as<br>
        mentioned the<br>
                          tutorial? Do I need to extend the sampling ?<br>
        or any other<br>
                          reason?<br>
<br>
                          Please help me.<br>
                          Thank you.<br>
<br>
                          -Aswathy<br>
<br>
<br>
<br>
                      --                       ---------------------------------------------------<br>
                      Dr. Jochen Hub<br>
                      Molecular Biophysics group<br>
                      Dept. of Cell &amp; Molecular Biology<br>
                      Uppsala University. Box 596, 75124 Uppsala, Sweden.<br>
                      Phone: +46-18-4714451 Fax: +46-18-511755<br>
                      ---------------------------------------------------<br>
<br>
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                  --     Aswathy<br>
<br>
<br>
<br>
<br>
               --         Aswathy<br>
<br>
<br>
           --     Dr Thomas Piggot<br>
           University of Southampton, UK.<br>
<br>
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        --         Aswathy<br>
<br>
<br></div></div><div class="im">
    --     ========================================<br>
<br>
    Justin A. Lemkul<br>
    Ph.D. Candidate<br>
    ICTAS Doctoral Scholar<br>
    MILES-IGERT Trainee<br>
    Department of Biochemistry<br>
    Virginia Tech<br>
    Blacksburg, VA<br></div>
    jalemkul[at]<a href="http://vt.edu" target="_blank">vt.edu</a> &lt;<a href="http://vt.edu" target="_blank">http://vt.edu</a>&gt; | (540) 231-9080<div><div></div><div class="h5"><br>
    <a href="http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin" target="_blank">http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin</a><br>
<br>
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-- <br>
Aswathy<br>
<br>
</div></div></blockquote><div><div></div><div class="h5">
<br>
-- <br>
========================================<br>
<br>
Justin A. Lemkul<br>
Ph.D. Candidate<br>
ICTAS Doctoral Scholar<br>
MILES-IGERT Trainee<br>
Department of Biochemistry<br>
Virginia Tech<br>
Blacksburg, VA<br>
jalemkul[at]<a href="http://vt.edu" target="_blank">vt.edu</a> | (540) 231-9080<br>
<a href="http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin" target="_blank">http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin</a><br>
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</div></div></blockquote></div><br><br clear="all"><br>-- <br>Aswathy<br>