(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)
Copyright Ross Walker 2008

Setting up an Advanced System
(including basic charge derivation)
By: Bryan Lelanda, David Paula, Brent Kruegera & Ross Walkerb
aDept. of Chemistry, Hope College*
bSan Diego Supercomputer Center, University of California, San Diego

2.1) Attachment Process for the Linker and Fluorescein

The linker serves as an anchor for fluorescein, enabling us to attach our fluorescent dye to the A5' end of the polyAT decamer. By doing so we are able to computationally simulate the probe linkage present in many laser experiments. We will use the Sirius visualization program (a tutorial on Sirius is here), although in principle you could use another program of your choice. You could potentially do this in xleap but it would be difficult. This step is required because there is no crystal structure of the dye, linker and DNA system available. Hence we have to build ourselves a PDB file with a suitable structure that we will ultimately load into leap when making the prmtop and inpcrd files. If a crystal/NMR structure of the system you want to simulate is available then it is often a simple matter of editing this PDB file as necessary and the majority of the approach discussed in this section would not be needed.

Since we optimized the fluorescein dye and linker molecules separately in the previous section, we will have to bond these two molecules together as depicted in the introduction (figure ii). Fortunately there are several tools available that allow us to manipulate our molecules and make the necessary changes. The Sirius visualization program enables us to superpose fluorescein's NH group (NME cap) on top of the NH group present in the linker, as well as the carbonyl groups present in both molecules. These "caps" can then be deleted and the fluorescent linker coordinates saved in the same reference frame, making it possible to bond these molecules together in xleap.

With all of this in mind, execute the Sirius program and load both of the optimized structures from the earlier charge fitting (floF_opt.pdb, ln5_opt.pdb). Select, Tools > Structure Browser to display the contents of the two files. This will come in handy, as it makes the selection of entire molecules and atom segments easy.

Figure 2.1

2.1.1) Using Sirius to Change the Coordinates of Fluorescein and Linker

In order to select the atoms we are going to superpose, we will need to move the linker away from the fluorescein molecule. To begin, "highlight" the linker molecule in the Structure Browser and select the "Set separate motion" button located in the toolbar.

The left mouse button rotates the selected atoms, while the middle button zooms, and the right button translates. At times it may be useful to rotate both molecules in order to gain a better perspective of the atoms. This can be accomplished by selecting the "Set common motion" button located in the toolbar, immediately below the set separate motion button pictured above. Note: Mac OSX users may not be able to view the button tooltips, as Sirius is still Beta. This bug should be fixed in later versions of the software. For a complete list of the Sirius buttons and what they do, Select Help > Sirius Help... and search for "toolbar" using the index tab.

Figure 2.2

Now that we are able to differentiate between the two molecules, we can superpose the caps and delete the extra atoms. Select, Structure > Structure superposition > atom anchor-based superposition. The following menu should appear (Figure 2.3).

You can use the mouse to select the atoms used in the superposition process. These atoms are selected in groups of two (one from the aligned structure, the other from the reference structure). For the purpose of this tutorial it does not matter which structure is the “aligned”, and which is the “reference”. Therefore, we have arbitrarily assigned the linker as the “aligned” structure. Make note that the first atom selected in each pair has to be from the aligned structure, and the second from the reference structure. Select the first atom N, from the linker, and the second atom N, from the dye. Repeat this selection process for the carbonyl atoms C and O, then press OK. The resulting structures should resemble those of Figure 2.4.

Superposition GUI After Sirius Superposition
Figure 2.3
Figure 2.4

2.1.2) Deleting the Remaining Optimization Caps and Saving the pdb file

Our combined fluorescein/linker molecule, which we will call FAM is almost complete. The next step will be the cap deletion as discussed earlier in the tutorial. The ACE and NME caps were only needed for charge derivation, and the extra atoms should not be present in the final FAM molecule. As you recall, the NME cap on fluorescein contains the following atoms: CH3NH (Figure 1.2). Highlight these atoms using the Structure Browser pane, and select Structure > Edit Structure Objects. A GUI should appear; make sure the "delete a set of atoms" radio button is selected, as well as the "currently selected atoms" button, and press OK. The above process should be repeated for the ACE cap present on the linker. The atoms present on the ACE cap are CH3CO (Figure 1.2). Finally, the phosphate capping group also needs to be removed from the linker. Select the P-O-O-O-CH3 atoms and delete. The structure should resemble that of Figure 2.5 after all extra atoms have been deleted.

Fluorescein/Linker after cap deletion
Figure 2.5

We have now successfully modified our molecules using Sirius, and are ready to move on to the next step in our tutorial. However, in order to make things run smoothly in the following sections, we will need to merge our structures into one pdb and edit the file so it can be correctly read into the xleap program. The naming convention found in the current version of Sirius does not allow for atom names above 3 digits, this poses a problem for research groups working with large molecules such as fluorescein. Second, the merged structures have two different residue names. We will be creating one library file that tells xleap how to recognize the fluorescein/linker construct, thus each atom in our pdb must be in the same residue and must have unique names. (Note in principle one could also set this up as two separate residues with two separate library files. The choice is yours.) Third, the connectivity information saved in the new pdb file is invalid, and will need to be deleted. Note that several of these issues will be fixed in later versions of Sirius, as it is still in Beta. Until then we will manually address these problems.

So, with that said, we can move on and merge our molecules together into one pdb. Sirius can automate this step for us and reduce the amount of text editing that needs to be done. To accomplish this task, select Structure > Edit selected objects. Make sure that you select the "merge two structures together" radio button. Next, we will use the provided drop down menus to select fluorescein as the first structure, and the linker as the second. Upon setting these variables we will name the new structure fam5, and click OK. After merging the structures, we can save the new pdb file using File > Save structure. fam5.pdb

2.2) Modifying the fam5.pdb file

In this section of the tutorial we will modify the newly created fam5.pdb file to make it compatible with xleap. This can be done in a text editor of your choice. The first thing we will edit in the tutorial is the atom names. Each atom has to possess a unique name. Because this is an advanced tutorial we will not go into detail about standard atom naming schemes for the pdb file format. Next, we will assign a residue name of FAM for the entire molecule, and change the linker's chain value of 2 to 1 so that it is considered to be part of the first residue. The final major change to the pdb will be deletion of the connectivity information that was provided by Sirius. The only data we want from the pdb file is the coordinates of our fluorescein and linker atoms. The final pdb after modifications should look like the following file: fam5_edit.pdb. We are now ready to load the edited pdb into xleap and start building our library file.


*Funding and computational support for the creation of this tutorial was provided by NSF-CIEG (BDI0726924), NSF-REU, NSF-MRI, HHMI and ACS-PRF.

(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)
Copyright Ross Walker 2008