Part 1: Molecular Visualisation

Comparing Trajectories

The final thing we will learn in VMD is how to load up and compare the output of two different molecular dynamics trajectories. Please download the below two files and place them into the same directory as h7n9.pdb and h7n9.dcd

Next, we will load up the h7n9_rk.pdb file in addition to the h7n9.pdb file that is already loaded into your VMD session. To do this, click “File | New Molecule…” in the VMD main window to open the “Molecule File Browser” window. Then, ensuring that the “Load files for:” selector is selecting “New Molecule”, click “Browse” and find the h7n9_rk.pdb file that you downloaded. Select this and click “Load” to load this file.

Image opening h7n9_rk.pdb

Next, we will load the trajectory in h7n9_rk.dcd into the molecules loaded from h7n9_rk.pdb. To do this, in the “Molecule File Browser” window change the “Load files for:” selector to select “2: h7n9_rk.pdb”. Then click “Browse” and find the h7n9_rk.dcd file that you downloaded. Select this and click “Load” to load this file.

Image opening h7n9_rk.dcd

The molecules in h7n9_rk.pdb are currently visualised using the “lines” representation. Go into the graphical representations window and add representations to match those for h7n9.pdb. To do this, you will need to ensure that the “Selected Molecule” selector at the top of the “Graphical Representations” window is selecting “2: h7n9_rk.pdb”, e.g.

Image opening h7n9_rk representations

Once you have done this, zoom in to get a view similar to that in the above picture. You should be able to see that the molecules in h7n9_rk.pdb are not aligned with those in h7n9.pdb, and so oseltamivir in h7n9_rk.pdb appears rotated by about 90 degrees compared to oseltamivir in h7n9.pdb. This makes comparison of the two trajectories difficult. What we need to do is to ensure that all of the frames from both trajectories are aligned against a common frame of reference. In general, the best frame of reference for any bimolecular alignment is the protein backbone at the start of the trajectory for one of the structures. In this case we will use the backbone of h7n9 neuraminidase.

To align the structures, we need to use the “RMSD Trajectory Tool” that comes with VMD. Open this by clicking “Extensions | Analysis | RMSD Trajectory Tool” in the VMD main window. This will open up the “RMSD Trajectory Tool” window, which is described in the image below.

Image of RMSD trajectory tool

We would like to align all frames of both h7n9.pdb and h7n9_rk.pdb against the first frame of h7n9.pdb. We want to align only the protein backbone, so we need to ensure that;

  1. The “Atom selection box” contains “protein”,
  2. That “Backbone” is ticked,
  3. That we have selected h7n9.pdb in the “Molecule list”,
  4. That “Selected” is chosen in the “Reference selector” and
  5. That the reference frame “ref:” is “0” in the “Frame selector”.

Once this is set, click “Align” to align the frames, and you should see something like this (you may need to rotate and zoom back into the molecule).

Image of aligned molecules

If you now play the movie you should be able to see that the binding position of oseltamivir in H7N9 and H7N9-R292K neuraminidase is slightly different. The mutation has changed the arginine (which was hydrogen bonding with the oxygen at the bottom of oseltamivir) into a lysine. The lysine is too short to hydrogen bond with the oxygen, so it is rotated away to the left. Here, it pushes away what is called the “bulky group” of oseltamivir, pushing that up and out of the binding site. You can see this more clearly by temporarily switching off the view of h7n9.pdb by double-clicking on the letter “D” next to h7n9.pdb in the VMD main window (the “D” stands for “Draw”, and double clicking it will toggle between “draw the molecule”, when the “D” is coloured black, and “hide the molecule” when the “D” is drawn red).

Image of bulky group

If this is not clear, try rotating and zooming in around h7n9.pdb and h7n9_rk.pdb while the movie is playing until you are happy that you can see that oseltamivir has been tilted up and slightly out of the binding site.

While this change in binding mode of oseltamivir can be seen in the movie, you would need to back up this qualitative visual observation with a quantitative measurement if you were going to report it in a paper. To do this, we will use the graphing tools introduced in the previous section to graph the distance between the oxygen of oseltamivir and the nitrogens of arginine 292 and lysine 292 from H7N9 and H7N9-R292K neuraminidase.

First, make sure that only h7n9.pdb is drawn.

Image of label compare

Once everything is set, click the “Graph…” button and you should see something like this;

Image of label comparison graph

This shows clearly that the oxygen-nitrogen distance in the mutant H7N9-R292K neuraminidase is much larger and more variable than that for the wild type. If you want, you can save these distances to a text file by using the option “File | Export to ASCII Matrix”. This can then be loaded into a spreadsheet package (e.g. Excel or LibreOffice) to produce a publication-quality graph.

On its own, this single oxygen-nitrogen distance is not enough to fully quantify the change in binding mode of oseltamivir. Have a look at the movie and try to come up with other distances (and angles) that will let you quantify the change that you can observe qualitatively in the movie. Once you have identified suitable distances and angles, use the above procedure to create graphs that would be of publication quality.

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