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<div class="" style="word-wrap:break-word; line-break:after-white-space">Hi everyone, here is one tutorial that those at U of Montana may want to use today as it was written specifically for use with the linux workstation that you will have access to. Those
at U of Lethbridge and those that are more familiar with VMD than Chimera will likely want to use a tutorial at
<a href="http://ambermd.org" class="">ambermd.org</a> that I will specify during class.
<div class="">Likely the biggest hurdle will be actually connecting to the linux workstations at U Montana and U Lethbridge, connecting is possible using a PC (using PuTTY
<a href="https://www.chiark.greenend.org.uk/~sgtatham/putty/latest.html" class="">
https://www.chiark.greenend.org.uk/~sgtatham/putty/latest.html</a>, or other methods) but will be easier from a Mac or Linux machine. We will use 30 minutes of class time today to get everyone connected and started on the tutorials.</div>
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<div class="">See you at 11am.</div>
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<div class="">Travis</div>
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Travis Hughes<br class="">
Associate Professor <br class="">
Biomedical and Pharmaceutical Sci.<br class="">
University of Montana, Missoula<br class="">
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<div class="">Thanks to Hughes lab Alumni Trey Patton for creating this note/tutorial: As the disclaimer states there may be some hiccups in this tutorial, but with Travis’s help we should be able to get it figured out.</div>
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<div class="">DISCLAIMER: This is still a work in progress, so use this at your own risk for the time being. For the most part, it's pretty complete. There are some issues that have come up throughout other runs that I have notes about fixing in the notes for
those runs. I would recommend searching my notebook for errors. Example: I got a PMEMD error during minimization. Search PMEMD in my notebook to find a fix. Other such errors may or may not be here.</div>
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<div class="">1.Save just the part of the pdb that you want to use</div>
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<div class="">paste in wt sequence that we generally use for simulations (obviously use mutant sequence if modelling mutant) and give the sequence a name at teh top (wt here) then hit OK:QLNPESADLRALAKHLYDSYIKSFPLTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLY</div>
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<div class="">associate the main pdb with your input sequence, hit ok</div>
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<div class="">missing amino acids in your structure are now highlighted in red boxes:</div>
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<div class="">click on moddellor loops/refinement</div>
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<div class="">click all missing structure and in general 1 model is good enough. You will need to sign up on the modellor website to get this part to work. Hit OK.</div>
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<div class="">Modellor does its thing and you get out a blue model overlayed on the original</div>
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<div class="">delete everything but the ligand and model. save this as a pdb, ready to go on to the next step, H++.</div>
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<div class="">The first step in preparing your ligand and protein for a simulation is to run modeller through chimera. Refer to<span class="x_Apple-converted-space"> </span><span class="" style="font-style:italic">"modelling in missing structure using chimera"</span><span class="x_Apple-converted-space"> </span>to
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<div class="">Once completed, we will be using two different sites to prepare our ligand and protein. </div>
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<div class="">Protein: H++</div>
<div class="">Ligand: Red Server</div>
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<div class=""><span class="" style="font-weight:bold">Protein Preparation using H++:</span></div>
<div class="">H++ sets protonation states of histidines, etc. The website to complete this step can be found at <a href="http://t.mail-svc.evernote.com/f/a/EGdHcKmcm6iE3eYWLI-MKg~~/AADd_wA~/RgRiUZRtP0QcaHR0cDovL2Jpb3BoeXNpY3MuY3MudnQuZWR1L1cDc3BjQgpgZ20Pb2BMENppUhp0cmF2aXMuaHVnaGVzQHVtb250YW5hLmVkdVgEAAAABg~~" shape="rect" class="" style="color:rgb(54,151,179); font-weight:bold; text-decoration:none; font-family:"Segoe UI"">http://biophysics.cs.vt.edu</a></div>
<div class="">You should set the pH to 7.4 (biological pH) and the salinity to 0.05 (0.05M)</div>
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<div class="">If you don't have an account created, register at the bottom left.</div>
<div class="">After logging in, go up to the left side of the screen and click "PROCESS A STRUCTURE"</div>
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<div class="">Upload your protein structure</div>
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<div class="">Click "Process File"</div>
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<div class="">After changing salinity (0.05) and pH (7.4), click Process..</div>
<div class="">H++ will remove your ligand, which will need to be put back into the protein after completing the Red Server Step</div>
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<div class="">For clarity, since I have run into this problem twice now:<span class="x_Apple-converted-space"> </span><span class="" style="text-decoration:underline">We will need to add the ligand back into the H++ pdb, run the PROTEIN and LIGAND through pdb4amber
(below), AND THEN extract the ligand from the pdb4amber pdb file to be used in the Red Server.</span> </div>
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<div class="">The second thing we should do is format our new H++ protein and Ligand file. You can open it up in any text editing software. I personally use Atom, but notepad or any of the others will work just fine. Amber has a problem with reading anything
that says MODEL or ENDMDL, so we will need to delete those from our pdb file (you can do a simple ctrl-f to find them). These will normally be at the end of the protein/ligand. A TER (terminate) line needs to be placed at the end of the protein information
(before the ligand), and at the end of the ligand. </div>
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<div class="">For example:</div>
<div class=""><span class="" style="font-family:"Segoe UI"">ATOM 4088 C GLU 251 21.050 11.720 7.820 1.00 0.00 C #protein text</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">ATOM 4089 O GLU 251 19.804 11.678 8.010 1.00 0.00 O #protein text</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">ATOM 4090 OXT GLU 251 21.776 10.705 7.638 1.00 0.00 O #protein text</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"; font-weight:bold">TER</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">HETATM 1 O37 965 252 50.160 16.409 -5.403 1.00 0.00 O #ligand text</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">HETATM 2 C35 965 252 50.401 16.325 -4.182 1.00 0.00 C #ligand text</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">HETATM 3 O36 965 252 51.511 16.576 -3.734 1.00 0.00 O #ligand text</span></div>
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<div class="">The first thing you will need to do is send the file to the workstation. To do that, use the following line in your bash command prompt (if you don't have bash, refer to "getting started with Amber" in Trey's notebook:</div>
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<div class=""><span class="" style="font-style:italic">scp (file name) (account name)@10.8.161.140:~/(destination path)</span></div>
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<div class="">This puts your file into the designated folder on workstation.</div>
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<div class="">Now we will be running pdb4amber using the following line:</div>
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<div class=""><span class="" style="font-style:italic">pdb4amber -i (name of input file) -o (name of output file)</span></div>
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<div class=""><span class="" style="font-style:italic">Example: pdb4amber -i chaina5ugm_h++_lig2.pdb -o 5ugmA.pdb</span></div>
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<div class=""><span class="" style="font-weight:bold">This file will be used later on so make sure you note which file was created using pdb4amber. An easy way to remember is to put pdb4amber in your output file name (or just write it down in your notebook)</span></div>
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<div class="">pdb4amber will give AMBER names to histidines (HIE, HIP, HID)</div>
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<div class="">To transfer the pdb4amber file back to your computer to extract the ligand, use the following line:</div>
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<div class=""><span class="" style="font-style:italic">scp (name)@10.8.161.140:~/path-to-pdb4amber-file /path-to-where-you-want-the-file</span></div>
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<div class=""><span class="" style="font-style:italic">Example. scp<span class="x_Apple-converted-space"> </span><a href="mailto:jon@10.8.161.140" class="" style="color:rgb(54,151,179); font-weight:bold; text-decoration:none">jon@10.8.161.140</a>:~/NMP422/build/NMP422_pdb4amber
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<div class="">****Might need to protonate ligand before running through pdb4amber****</div>
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<div class=""><span class="" style="font-weight:bold">Preparing your Ligand with Red Server:</span></div>
<div class=""><span class="" style="text-decoration:underline">THIS IS DONE AFTER RUNNING THE H++ PROTEIN AND LIGAND THROUGH PDB4AMBER!!</span></div>
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<div class="">To complete this step, you will go to this website: <a href="http://t.mail-svc.evernote.com/f/a/GmcazYhaB2k7Msek2wt3Jw~~/AADd_wA~/RgRiUZRtP0Q7aHR0cDovL3VwanYucTRtZC1mb3JjZWZpZWxkdG9vbHMub3JnL1JFRFNlcnZlci1EZXZlbG9wbWVudC9XA3NwY0IKYGdtD29gTBDaaVIadHJhdmlzLmh1Z2hlc0B1bW9udGFuYS5lZHVYBAAAAAY~" shape="rect" class="" style="color:rgb(0,0,238); font-weight:bold; text-decoration:underline">http://upjv.q4md-forcefieldtools.org/REDServer-Development/</a></div>
<div class="">It is much easier to make an account and go from there.</div>
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<div class=""><span class="" style="font-family:"Segoe UI"">My computer had an issue with Red Server. It kept saying Java and Cookies weren't enabled. I went into my browser's settings (Chrome: Settings --> Advanced --> Content Settings --> Java & Cookies -->
Allow --> Type in a url). Allow </span><a href="http://t.mail-svc.evernote.com/f/a/TJwJshLcpzRI_6XCVUDfkQ~~/AADd_wA~/RgRiUZRtP0QlaHR0cDovL3VwanYucTRtZC1mb3JjZWZpZWxkdG9vbHMub3JnL1cDc3BjQgpgZ20Pb2BMENppUhp0cmF2aXMuaHVnaGVzQHVtb250YW5hLmVkdVgEAAAABg~~" shape="rect" class="" style="color:rgb(54,151,179); font-weight:bold; text-decoration:none; font-size:13px; letter-spacing:normal; orphans:2; text-indent:0px; text-transform:none; white-space:nowrap; widows:2; word-spacing:0px; font-family:Roboto,"Segoe UI",Tahoma,sans-serif">http://upjv.q4md-forcefieldtools.org</a></div>
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<div class="">The first step to preparing our ligand is to prepare it for the Red Server. You do this by protonating the molecule. We can do this using chimera.</div>
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<div class="">965 is my ligand. I am going to select it and then invert selection to select everything BUT the ligand.</div>
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<div class="">Delete everything but the ligand</div>
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<div class="">Now we are going to add Hydrogens to the ligand. Make sure you look at the ligand's structure to estimate whether certain functional groups will be protonated at biological pH. Remember: we can look at the pKas of functional groups to make this
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<div class="">I just leave all of these settings the way they are when adding hydrogens. If it doesn't work the first time, try selecting the molecule.</div>
<div class="">Protons added</div>
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<div class="">Save the molecule as a pdb</div>
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<div class="">In order to run on the Red Server, there are a few more things we need to do. First, we need to make a project.config file. Red Server describes how to do this on their website, but I will try to explain it as well.</div>
<div class="">The file is as follows:</div>
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<div class=""><span class="" style="font-family:"Segoe UI""># A well defined title</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">MOLECULE1-TITLE = GW3965 <--- Molecule name</span></div>
<div class=""><span class="" style="font-family:"Segoe UI""># DHA</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">MOLECULE1-TOTCHARGE = 0 <--- charge of the molecule at biological pH (7.4)</span></div>
<div class=""><span class="" style="font-family:"Segoe UI""># With MOLECULE1-SPINMULT = 1 the default option is kept (not useful)</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">MOLECULE1-SPINMULT = 1</span></div>
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<div class="">This molecule has a carboxylic acid group that will be charged, and the tertiary nitrogen should have a charge. The total charge of the molecule should be zero.</div>
<div class="">Also, here is </div>
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<div class="">The name of the ligand is specific for the zip file we are going to make to run on the Red Server. The zip file will include the ligand and the profile.config file.</div>
<div class="">Red Server requires the name Mol_red1n.pdb for the ligand file. There are specific criteria that matches the name, and Red Server explains that.</div>
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<div class="">Now that we have the zip file created, we can send it to the Red Server.</div>
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<div class="">Login on the next window</div>
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<div class="">Follow the next screen until you need to give a project name and name it your ligand name. Change nothing else. The next screen will be to upload your zip file. Do that.</div>
<div class="">After uploading the molecule, click "Let's Go!" and your run will start. Red Server can take a while. Just depends on the server capacity for that given time.</div>
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<div class="">Once your Red Server run completes, you will receive a link to get your files. We only need the mol2 file.</div>
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<div class="">The download button will open to a text file. Simply copy and paste it into a text editing program (I use Atom) and save it as a mol2.</div>
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<div class=""><span class="" style="font-weight:bold">Antechamber</span></div>
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<div class="">Now we will need to transfer our .mol2 ligand to the workstation using the same line we used to transfer the protein/ligand (scp ....) to run Antechamber.</div>
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<div class="">The line for antechamber is as follows:</div>
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<div class=""><span class="" style="font-style:italic">antechamber -i (ligand.mol2 file) -fi mol2 -o (ligandgaff.mol2) -fo mol2 -at gaff2</span></div>
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<div class=""><span class="" style="font-style:italic">Example: antechamber -i Mol-sm_m1-c1_eda.mol2 -fi mol2 -o edagaff.mol2 -fo mol2 -at gaff2</span></div>
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<div class="">This step gives gaff names to our ligand's atoms.</div>
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<div class=""><span class="" style="font-weight:bold">Making frcmod files</span></div>
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<div class="">We will need to use an frcmod file to create our parameter and coordinate files. The line to do so is as follows:</div>
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<div class=""><span class="" style="font-style:italic">parmchk2 -i (gaff file) -f mol2 -o (gaff file.frcmod) -s gaff2</span></div>
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<div class=""><span class="" style="font-style:italic">Example: parmchk2 -i edagaff.mol2 -f mol2 -o edagaff.frcmod -s gaff2</span></div>
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<div class=""><span class="" style="font-weight:bold">tleap + molarity.perl</span></div>
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<div class="">tleap will allow us to make topology/parameter and coordinate files for our runs</div>
<div class="">Here we will be using a script to do this: </div>
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<div class=""><span class="" style="font-family:"Segoe UI"">#!/bin/bash</span></div>
<div class=""><span class="" style="font-family:"Segoe UI""># load script using: tleap -s -f script-name.tleap</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">source leaprc.protein.ff14SB</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">source leaprc.gaff2</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">source leaprc.water.tip3p</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">loadAmberParams<span class="x_Apple-converted-space"> </span><span class="" style="font-family:"Segoe UI"; font-weight:bold">(your frcmod file you just created)</span></span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">BRL= loadMol2<span class="x_Apple-converted-space"> </span><span class="" style="font-family:"Segoe UI"; font-weight:bold">(your gaff file) ****BRL is meant for the Ligand name. Apparently sometimes
AMBER doesn't like numbered ligand names, so you can manually change them to LIG. Just make sure your variable (BRL) is set accordingly. For example, if you want to use BRL, make sure your ligand is labeled BRL in the text files)</span></span></div>
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<div class=""><span class="" style="font-family:"Segoe UI"">complex = loadpdb S-protlig.pdb<span class="x_Apple-converted-space"> </span><span class="" style="font-family:"Segoe UI"; font-weight:bold">(replace your pdb4amber created complex with S-protlig.pdb)</span></span></div>
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<div class=""><span class="" style="font-family:"Segoe UI"">check complex</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">charge complex</span></div>
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<div class=""><span class="" style="font-family:"Segoe UI"">addions complex Na+ 0</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">addions complex Cl- 0</span></div>
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<div class=""><span class="" style="font-family:"Segoe UI"">solvateoct complex TIP3PBOX 10.0 iso</span></div>
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<div class=""><span class="" style="font-family:"Segoe UI"">addions complex K+ 0<span class="x_Apple-converted-space"> </span><span class="" style="font-family:"Segoe UI"; font-weight:bold"><-- set this to zero for this run. We will be running another script
to find out the additions after running tleap for the first time.</span></span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">addions complex Cl- 0 <--<span class="x_Apple-converted-space"> </span><span class="" style="font-family:"Segoe UI"; font-weight:bold">set this to zero for this run. We will be running another script
to find out the additions after running tleap for the first time.</span></span></div>
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<div class=""><span class="" style="font-family:"Segoe UI"">saveamberparm complex S-rosi.prmtop S-rosi.inpcrd</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">savepdb complex S-rosisolv.pdb</span></div>
<div class=""><span class="" style="font-family:"Segoe UI"">quit</span></div>
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</div>
<div class=""><span class="" style="font-weight:bold">There are certain errors that will show up in tleap. I have gotten fatal errors because I missed a single, capitalized letter in a ligand atom. If you run into "Fatal: .......does not have an atom type"
that your pdb4amber protein pdb, red server ligand pdb, mol2 file, and gaff file all have the same exact atom names in them.</span></div>
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<div class="">Now that we have run tleap for the first time, we will need to run a script called molarity.perl: </div>
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<div class="">This script is a little more difficult to run. tleap has just created an inpcrd file and you will need to take the last line of that file. Once you have it, you can run the molarity.perl script using: </div>
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<div class="">perl molarity.perl 0.05 X X X X X X (The Xs are the six columns of values from the inpcrd file)</div>
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<div class="">So an example is: </div>
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<div class=""><span class="" style="font-family:"Segoe UI"">perl molarity.perl 0.05 </span><span class="" style="font-family:"Segoe UI"">81.8353170 81.8353170 81.8353170 109.4712190 109.4712190 109.4712190</span></div>
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<div class=""><span class="" style="font-family:"Segoe UI"">Now edit your tleap file to include the K+ and Cl- ions your molarity.perl script just gave you, and rerun tleap.</span></div>
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<div class=""><span class="" style="font-weight:bold">Minimization and Equilibration:</span></div>
<div class="">The next step is to minimize and equilibrate your system using the following script: </div>
<div class="">There are two edits that need to be made, and that is inserting your prmtop and inpcrd files into the script.</div>
<div class="">After editing, transfer the file to the workstation and run it using ./mesmy.sh</div>
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<div class=""><span class="" style="font-weight:bold">Hydrogen Mass Repartitioning:</span></div>
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<div class="">We will need to run a hydrogen mass repartitioning once our minimization and equilibration is complete. </div>
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<div class="">on workstation type:</div>
<div class="">parmed nameofyourfile.prmtop</div>
<div class="">HMassRepartition</div>
<div class="">outparm nameofyourfilehmr.prmtop</div>
<div class="">quit</div>
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<div class="">This will create a hydrogen mass repartitioning file that we will use in our run.</div>
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<div class=""><span class="" style="font-weight:bold">Running the simulation:</span></div>
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<div class="">You will need three other files for this: con.in, new.in, and run.sh</div>
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<div class="">For whatever reason, I couldn't attach an example of con.in. Just simply copy the file into your folder that contains your build, a, b, etc. files</div>
<div class="">For example, my con.in is in /storage/trey/NMP422, not in /storage/trey/NMP422/a or b or build</div>
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<div class="">new.in needs to be in the same place as con.in. The new.in file gives reaction conditions to your simulation.</div>
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<div class=""></div>
<div class="">run.sh will need to be in your build directory. For example, mine is in /storage/trey/NMP422/build. This file is the script that allows you to start the simulation.<span class="x_Apple-converted-space"> </span><span class="" style="font-weight:bold">DON'T
DO THIS YET,</span><span class="x_Apple-converted-space"> </span>but to run the script type "./run.sh". You will need to edit this file at some point to put your simulation onto a specific gpu. The line to do this is:</div>
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<div class="">export CUDA_VISIBLE_DEVICES=2 #gpu 0-3</div>
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<div class="">Choose between gpu 0-3 to run your simulation. You can check to see which gpus are available by typing in nvidia-smi. This will open a dialogue box that tells you which are free. Just look at the Memory-Usage section, and choose one that says
"0MiB/8113MiB"</div>
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</div>
<div class="">To start your simulation and have it run even when you are off the workstation, you will need to open up a virtual screen. Type "screen" into the command prompt. This will open up a new screen that has you in the same directory. From here, we
will need to run the run.sh script to start the simulation. This starts your simulation. The following are important key presses to either keep the simulation running or cancel it:</div>
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</div>
<div class="">ctrl-a-c to terminate run and close screen</div>
<div class="">ctrl-a-d to close screen but have run continue</div>
<div class="">type screen to open up new window to begin run -- screen opens up new window that persists even after closing computer/putty. To kill you need to be in the screen and type "exit"</div>
<div class="">Can also hit "ctrl-a" once and then hit "k" to kill it.</div>
<div class="">If you only have one screen open you can type "screen -r" to get back to it. If you have multiple screens "screen -r" will show you all open screens.</div>
<div class="">To go back to an open screen, type "screen -r" to see which screens are open. You will see something like "screen 6404.(text)." To go back to that screen, type "screen 6404" or whatever number is there. This should take you back to the screen.</div>
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<div class="">Your simulation is now running! Congratulations!</div>
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