Cas12a Variant Visualization

In the previous slide we plotted our MSA alignment, the pLDDT scores, and the predicted alignement error. However, it is also useful to visualize the actual predicted protein structure and compare it to the known structure if there is one. Here we use a software called PyMOL to do just that:

Here we see that PyMOL takes either the PDB ID or a PDB file and creates a vizualization for us to examine. If you have not done so already please download PyMOL and open the app. You should see a window like the follwing:

Here we have a:
History Window with log of previous commands
Command Interface to enter PyMOL commands
List of Objects Loaded which list of objects/proteins that have been loaded into PyMOL
Visualization Window to visualize protiens loaded into PyMOL
Let's try on our data!

Download AlphaFold Output

First we will need to download our predicted structure pdb file for the mutant, Cas12a mut2-CWF. To this, go to Files > Home Directory:

Now, click on cas12a_af2 and download the following file, mut2cwf.pdb:

Importing Structures

To visualize this protein structure in PyMOL, open PyMOL on your computer
Go to File > Open - then choose your pdb file.
The file we have loaded is the Cas12a-CWF mutant, let's fetch the structure for the wild type Cas12a protein:

fetch 5xus

We will now align this structure with the Cas12a-CWF mutant. So in the PyMOL command prompt enter:

align mut2cwf, 5xus

Viewing/Coloring Structures

To view one structure at a time, you can use the disable command to hide one of the structures:

disable 5xus

To see this structure again we can simply use the enable command:

enable 5xus

To change the color of our structures we can use the following command:

color lightblue, 5xus

color salmon, mut2cwf

Visualizing Variants

Now that we have aligned/colored our structures, let's select residues on the RuvC Domain on the Cas12a mut2-CWF and the Cas12a wild-type:

select resi 863+952+965+1214 and name CA

Note

Note we are only selecting the alpha carbons so that when we label these residues we only have one label per residue

To label these residues we can use the following:

label sele, " %s%s" % (resn,resi)

With these residues selected we can color them to visualize them easier:

color red, sele

Let's now zoom into this region:

zoom sele

To capture this image we can go to File > Export Image As > PNG... > Save PNG image as ... and enter a name for your image!
Alternatively, you can take a screen shot and save.

Can you spot any disordered regions that AlphaFold2 may not have predicted well? Include an image displaying one of these regions.

Ma et al. 2022 note that the Cas12a mut2-CWF has a more open active site than the wild type Cas12a. Disable the Cas12a mut2-CWF mutant and include an image of the wild type Cas12a. Then enable Cas12a mut2-CWF mutant, disable the wild type Cas12a and include an image of the Cas12a mut2-CWF mutant. Do you agree that the catalytic site appears more open in comparison to the wild type structure (5xus)?