Immunological 3D printing

Part 1: the pictures

As a good little geek, I’ve been itching to have a play with 3D printers for a while now. At first I’d mostly contemplated what bits and bobs I might produce for use in the lab, but then I started to see a number of fantastic 3D printed protein models.

Form is so important to function in biology, yet a lot of the time we biologists forget or ignore the shape and make-up of the molecules we study. As Richard Feynman one said, “it is very easy to answer many of these fundamental biological questions; you just look at the thing”.

3D printing protein structures represents a great opportunity to (literally) get to grips with proteins, cells, microbes and macromolecules. While I still recommend playing around with PDBs to get a feel for a molecule, having an actual physical, tactile item to hold appeals to me greatly.

So when I got access to the UCL Institute of Making, I got straight to work printing out examples of the immune molecules I study, T-cell receptors. You can read about how I made them here. Or, if you're just here for some pretty pictures of 3D prints, continue; here are the two I've printed so far.

Here are the two finished products! I apologise for the quality: a combination of my garish fluorescent office lighting and shonky camera-phones does not a happy photo make.

My first try: 3WPW. This is the A6 TCR, recognising the self-peptide HuD bound in the groove of the class I HLA-A2. HLA-A2 is coloured in dark pink, with β₂ microglobulin in light pink, while the alpha and beta TCR chains are shown by light and dark blue respectively.

I particularly love the holes, crevices and caves pitted throughout the molecules. Having spent a goodly deal of time painstakingly pulling the scaffolding material out of these holes, I can confirm that you do indeed get a feel for the intricate surfaces of these structures.

You can imagine the antigen presenting cell on the left, with the T-cell on the right, as if we were watching from within the plane of the immunological synapse.

As a brief aside, in playing around with the 3PWP structure in PyMol (as detailed in an earlier blogpost) I was surprised to see the following; despite being a class I MHC (the binding grooves of which should be closed in at both ends) we can see the green of the peptide peeking out contributing to the surface mesh.

There's that HuD peptide!

The new addition: 3T0E. This brilliant ternary structure shows another autoimmune TCR, this time recognising a class II MHC, HLA-DR4, with an additional coreceptor interaction; enter CD4! Here we have the TCR chains coloured as above, while the HLA-DR alpha and beta chains are red and magenta respectively. Leaning in to touch the (membrane-proximal) foot of the MHC is the yellow CD4. Note that I took feedback, and this time went for a colour that didn't look so rice-puddingy.

The structure that became my second print was a particularly lucky find, as it contains not only a TCR-pMHC interaction, but also the CD4 coreceptor. This shot is angled as if we're inside the T-cell looking out across the synapse. If you imagine the various components of CD3 clustering around the constant region of the TCR you can really start to visualise the molecular complexity of even a single TCR-pMHC ligation event.

It's also quite nice to see that despite the differences in HLA composition between classes (one MHC-encoded chain plus B2M in class I versus two MHC-encoded chains in class II), they structurally seem quite similar by eye - at least at a surface level scale.

There you have it, my first forays into 3D printing immunological molecules. Let me know what you think, if you have any ideas for future prints - I'm thinking probably some immunoglobulins for the next run - or if you're going to do any printing yourself.

Part two: the how to.

5 minute PyMOL tutorial

I'm very lucky to have a pretty great PhD project, deep-sequencing T cell receptor repertoires. It's an interesting, exciting field, that I have only one major complaint about - I don't get to make any pretty pictures.

It sounds like a minor complaint, but it makes a difference when it comes to presentation time. My main output is sequence data; at best I produce some nice graphs, which rather lack the the impact of say, some beautiful confocal microscopy.

In order to get nice pictures to open a presentation on, I like to just fire up some molecular visualisation software and get some nice screenshots of whatever molecules are relevant to the talk. In case others wanted to do similarly - make nice pictures, without needing to become an expert in molecular visualisation or modelling - but didn't know how, here's a mini-tutorial as to how I do it.

First off, you need some visualisation software. If you're a student or teacher, I think you can get the excellent PyMOL for free, which I'll use in this blog, although there are others. I personally cut my visualisation teeth on a combination of Jmol, and (the now decidedly retro) RasMol - what you learn in one is broadly applicable to the others.

Let's get a structure to work on - head on over to the Protein Data Bank and look up something you're interested in. You can search by molecule name, ID (if you have it, you can find these in structural papers) or author name.

We need to download the PDB file for a molecule of interest, which is basically just a text file containing a set of x, y, z coordinates for all the atoms in that molecule (as described by solving the crystal structure, for example).

I've downloaded the PDB for 1MI5, a crystal structure for the LC13 TCR bound to HLAB8 complexed with a peptide from the EBNA 3A molecule of Epstein Barr Virus.

Fire up PyMOL - there's a two windows, a viewer and an interface - and open your PDB (note that if you already know the ID of your molecule, you can use this to open the file without downloading the PDB with the fetch command, e.g. 'fetch 1zgl').

At first it'll probably look like a big mess.

Orient yourself. It's pretty easy to move about with mouse; it's just hard to remember which button does what. Handily, they designers provided a nice little reminder in the bottom ride hand side.

Basically, left mouse button rotates, middle button translocates, and right mouse button zooms. Have a play, get used to it, it probably doesn't matter much anyway - if you're anything like me you'll forget it every time and have to rediscover it every time.

Now let's make it look pretty.

By default, all molecules are selected. We can use the five buttons in the top right to perform various tasks on whatever's currently selected.

The left column represents the different selections. The buttons on the right are the tasks that can be applied to those selections. Left to right: Action; Show; Hide; Label; Color

Unless I'm looking at specific interactions, I like cartoon display, as I think this makes it easiest to see generally what goes where in a structure, particularly if there's more than one polypeptide present. Click 'S' for show, then choose cartoon.

It looks somewhat fuzzy now, as it's showing the cartoon display overlain on everything else that was there. To get rid of all the fluff, go the 'H' (hide) menu, and hide the lines (default layout) and the waters (sporadic red dots).

Much better.

Now we want to be able to tell our different molecules apart. This is where the PDB web entry for our molecule really comes in handy, as it tells us what letter is used to refer to each separate molecule as. This allows us to make different selections, which we can then use to apply actions to particular molecules or subsets of molecules.

So, looking at our entry, I can enter the following code into the command line area of the interface window to generate our selections.

 select MHC, (chain a)  
 select B2m, (chain b)  
 select peptide, (chain c)  
 select alpha, (chain d)  
 select beta, (chain e)  

TCR-people, note that also for the three or four other TCR-pMHC complexes I've tried this on, they've all used the same letters for each chain, so this might be the convention.

You can then use the 'C' color button on each selection to colour them separately. I've arranged the molecule so you see down the groove of the MHC molecule, as this shows the interaction site of the whole complex well.

However, I always think it's a bit hard to see the peptide in amongst all the other molecules, so I've gone back and changed it to show as spheres.

After that, we just need to save a high-resolution image, which we can get through ray tracing:

 ray 2400, 2400  

Just save the image after ray tracing. I then cropped this down using Gimp.

All done!

When using crystal structures that you didn't produce, remember to give proper attribution; either cite the original paper, or provide the PDB ID.

For a more in-depth understanding of PyMOL, why not read the manual or check out the PyMOL wiki.