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Subsections

Chirality in proteins and nucleic acids

Introduction

All amino acids but glycine have at least one chiral center at C $_{\alpha }$ (see Fig. 1). Threonine and isoleucine have an additional chiral center at C $_{\beta}$ . According to the D-/L- naming convention, naturally occurring amino acids are found in the L-configuration. Note, however, that D-amino acids do occur in biology, e.g., in cell walls of bacteria. Nucleic acids also have chiral centers. For example, in DNA the atoms C1', C3', and C4' are chiral, while RNA has an additional chiral center at C2'. Chirality is central to all molecular interactions in biological systems. A simple experiment demonstrates the principle: try to shake someone's left hand with your right.

**Figure 1:** Chirality at the C $_{\alpha }$ carbon atom of amino acids. The two enantiomers are mirror images of each other, like the left and the right hand. Source: http://web99.arc.nasa.gov/ãstrochm/chirality.jpg
$\begin{figure}\begin{center} \par \par \latex{ \includegraphics[scale=0.5]{FIGS/chirality} } \end{center} \end{figure}$

Checking stereochemistry of the structure

To demonstrate the usage of the plugin we will start by checking a structure consisting of the protein EF-Tu in complex with a tRNA $^{\mathrm{Phe}}$ . The used structure is based on the the PDB structure 1OB2 in which errors have been introduced manually.

Currently, the chirality plugin can be used from the text console and through a GUI. The available commands provided by the pugin can be obtained by typing chirality on the Tk Console or the VMD console. The following information should be printed in the console:

Commands:
  check    -- identify chirality errors
  list     -- list identified chirality errors
  minimize -- fix chirality errors using energy minimization
  move     -- move hydrogen atom to fix chirality
  reset    -- reinitialize plugin state
  restrain -- generate NAMD extrabonds file to prevent chirality changes
  show     -- visualize identified chirality errors

In the same way, the usage information for any of the provided commands can be obtained. For example, typing chirality restrain will show the syntax of the command that generates restraints for the impropers at chiral centers which can be used in NAMD.

In the following, we will describe how to use the chirality plugin through its GUI, but all of the tasks can also be accomplished from the console.

1: Load the files chir_testcase.psf and chir_testcase.pdb into a new session of VMD.

2

Open the chirality GUI by selecting Extensions $\rightarrow$ Modeling $\rightarrow$ Fix Chirality Errors in the VMD Main menu. In the upper part of the GUI (see Fig. 2) the user can specify the molecule and the atom selection to be tested.

**Figure 2:** The chirality GUI window.
$\begin{figure}\begin{center} \par \par \latex{ \includegraphics[scale=0.4]{FIGS/chirality_basic} } \end{center} \end{figure}$

3

The button Check structure initializes the test. Identified unusual chiral center configurations are displayed in the Identified chirality errors form (see Fig. 3). In the present case there are 9 chiral errors identified in both the protein and RNA parts of the loaded structure.

**Figure 3:** Identified unusual chiral center configurations in the chirality GUI window.
$\begin{figure}\begin{center} \par \par \latex{ \includegraphics[scale=0.4]{FIGS/chirality_errors} } \end{center} \end{figure}$

4

By selecting an entry in the Identified chirality errors form and hitting the button Show selected chiral centers it is possible to visually inspect all the individual identified unusual chiral centers. In the created representation (see Fig. 4) the chiral center is highlighted by a transparent purple sphere, while the four bonded atoms are shown in CPK.

**Figure 4:** Representation of the chiral error in U12 of the tRNA
$\begin{figure}\begin{center} \par \par \latex{ \includegraphics[scale=0.4]{FIGS/chirality_show} } \end{center} \end{figure}$

Correcting chirality

1: If the shown chiral center has the wrong chirality, the user can tag an atom to be moved to flip the chirality at the selected center. This is done by hitting the button hydrogen. Note, currently only the moving of a hydrogen atom at a chiral center is supported. If the chirality at the displayed center is correct, nothing needs to be done. It is also possible to untag a tagged atom by selecting it and hitting none.

2: Once a chiral center has been inspected, and the atom to be moved has been tagged, the actual moving of the atom can be executed by hitting the button Move tagged atoms for selected chiral centers.

3

Since the described procedure (simple moving of the hydrogen atom) generates an unphysical geometry of the molecule, it is necessary to optimize the structure using an MD force field. The chirality plugin uses the AutoIMD plugin for this purpose. This final step is accomplished by selecting the chiral centers which should be relaxed and hitting the button Minimize/equilibrate selected chiral centers. This will open the AutoIMD Controls window (see Fig. 5).

**Figure 5:** AutoIMD window called by `chirality` plugin.
$\begin{figure}\begin{center} \par \par \latex{ \includegraphics[scale=0.4]{FIGS/autoimd_basic} } \end{center} \end{figure}$

4: Select Settings $\rightarrow$ Minimization Mode in the AutoIMD Controls window and hit then the Submit button. Once the AutoIMD session started, hit the Connect button and a minimization will start. Finish the simulation when the structure relaxed. Usually, it takes up to a few thousand steps until atoms do not move anymore. Is the structure minimized, hit the Finish in the AutoIMD Controls window. For more details about using the AutoIMD plugin, the user is referred to the AutoIMD user's guide
http://www.ks.uiuc.edu/Research/vmd/plugins/autoimd/ .

5: WARNING: Depending on the quality of the initial structure, it can happen that not all errors can be fixed in one run. Thus, it is important to check the final result again and correct the remaining errors, if necessary. In order to check the structure after the performed minimization/equilibration using AutoIMD, the chirality plugin should be reset. This can be done by hitting the Reset chirality plugin button at the bottom of the chirality window. Note, in the current implementation it will be necessary to select the molecule again in the top part of the chirality window. Note also that if the molecule to correct is solvated, it may be necessary to equilibrate the minimized parts of the structure. This can be done by selecting Settings $\rightarrow$ Equilibration Mode and submitting a new AutoIMD run.

6: Finally, it is a good advice to minimize the structure in the very last step. This should be done by selecting Settings $\rightarrow$ Minimization Mode in the AutoIMD Controls window, which is still open from the last AutoIMD session, and hit then the Submit button. As mentioned in the introduction, additional restraints during the correction of chirality errors. The restraints will be used always when AutoIMD is started from the chirality window. The equilibrium values for these restraints are very approximate and bring the configuration in the correct region. Thus, in order to obtain a optimized structure, it is necessary NOT to use the restraints defined by chirality. This is achieved by reusing the AutoIMD window from the previous minimization/equilibration; the restraints will not be taken into account.

7: Once all minimization/equilibration steps are performed, the corrected structure should be saved. This can be done in the following way. Select the molecule in the VMD main window. Create a representation containing all atoms in the Graphical Representations window. Select File $\rightarrow$ Save Coordinates in the VMD main window. Select all in the Selected atoms and hit Save. Finally, specify the file name in the Chose filename to save trajectory and hit OK.

Preserving stereochemistry during MD simulation

As mentioned in the introduction, structure optimizations and MD protocols applying external forces can introduce chirality errors into the simulated structure. To preserve stereochemistry, the chirality plugin offers the possibility to generate restraints in form of extrabonds which can be used in a NAMD simulations. This feature currently is available only from the console.

1: Assuming the structure of interest is loaded in the top molecule in VMD and one wishes to obtain restraints for all chiral centers in proteins and nucleic acids, the command reads
chirality restrain -o chirality-extrabonds.txt
The file chirality-extrabonds.txt contains now two improper restraints for each chiral center. One includes the chiral center and its non-hydrogen binding partners. In the second restraint, the chiral center is replaced by the hydrogen bound to it. The equilibrium values of the two impropers are set to the values present in the used structure.

WARNING: Note, the specified restraints modify the potential energy function of the system, and thus should not be used in equilibrium production runs. Rather, these restraints are supposed to be used only in simulations in which large forces occur due to poor initial geometry or due to externally applied forces, e.g., MDFF or TMD. Note also that although the final structure resulting from such simulations will be correct, the behavior of the system during the simulation is artificial.

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