Here at Biology in 3D, we often talk about the aesthetic side of structural biology and the power of structural images to inspire both artists and scientists. In the last Structure issue of 2016, we published a Letter to Editor by Shuguang Yuan, H.C. Stephen Chan, Slawomir Filipek, and Horst Vogel that emphasizes the importance of user-friendly structural biology data visualization strategies.

I invited the authors to say a bit more about what they are trying to accomplish and how they are trying to accomplish it, and to share some examples with us here at CrossTalk. Here, they discuss how they combined the tools PyMOL and Inkscape to create effective visual models.

Over the last few decades, our ability to solve structures of biomolecules and biomolecular complexes has grown at a fantastic rate, making structural biology information ubiquitous and relevant to very broad communities of life and chemical sciences. Most of the users of structural biology information begin and end their structural explorations via a visual inspection of 3D structural models. Interacting with the 3D models, usually via software, is one of the most basic and widespread methods we employ to understand the biological function of macromolecules and learn from the structures.

And yet we've heard many concerns about how counterintuitive and user-unfriendly some visualization tools are. This prompted us to write our Letter to Editor and put together two examples on how we combined PyMOL and Inkscape to prepare biological images with ease, in a clear, professional, and artistic way.

We won't go into details on PyMOL, as there are many helpful online resources for anyone interested in using this software (see here, here, here, and here). The same goes for Inkscape, a free program supported by a large group of users, video and other tutorials, and a gallery of fabulous examples (here, here, and here) as well as an active wiki. Instead, we want to focus on two examples of how we used the combination of these two pieces of software to create illustrations of our favorite protein, 5-HT 3 receptor (5-HT 3 R), and its ligand, serotonin.

Example 1. The 2D/3D hybrid diagram of protein-ligand interactions

Here, we use serotonin to illustrate the key steps in the process of going from coordinate files to the final 2D/3D diagram that depicts details of how a small-molecule ligand engages the protein. There are two key sources of experimental coordinate files for small-molecule ligands, the Protein Data Bank (PDB) and Cambridge Structural Database. If the PDB coordinates are your starting point for manipulating a small molecule, then you'll have to get rid of the macromolecule first. The most straightforward way of doing this is to do some minor text-based editing of the PDB file to retain only information on the small-molecule coordinates before importing them into PyMOL.

When using PDB coordinates as a source for your small-molecule ligand structures, it is important to remember that it is very likely that they will not contain information on hydrogen atoms. So adding hydrogen atoms back is the critical step in your ligand coordinate preparation, and PyMOL makes this easy to do via clicking on the object and following the prompts. One point to keep in mind is that PyMOL will add the correct number of hydrogen atoms to satisfy the neutral valency of individual atoms, which means that you'll have to inspect your small molecule and manually assign any charged states by hand. In our case, serotonin is a primary amine, and we manually assigned a +1 charge for this nitrogen (N) atom to make our serotonin ready for the next step.

We start building our 2D/3D diagram by focusing on the 3D object first, which is serotonin. Our objective here was to create a 3D representation of serotonin where atoms are represented as spheres and colored based on their element. To do this, we use PyMOL options that allow you to select the object and color by atom using preset color selections. In our case, carbon atoms are yellow, nitrogen is in blue, and oxygen is in red. Next, we displayed all the atoms in serotonin as spheres, and PyMOL made customizing the appearance and size of the spheres easy via command line. For those who want to make molecules more stylish, you can play around with the surface texture and sphere style. For example, we adjusted the light on our object and obtained a transparent background image, resulting in a high-resolution image.

From here, we went into Inkscape to combine the image we generated in PyMOL with the 2D diagram of the macromolecule, in this case 5-HT 3 R. The 2D/3D hybrid diagram that works really well to highlight key points of contact between a small molecule and the protein is one that places the small molecule in the center and surrounds it with protein structural elements that engage with it. Inkscape offers a large variety of tools for freehand drawing and creating custom-made lines and objects. It's intuitive and straightforward as well as very flexible.

For example, using Inkscape, we were able to surround serotonin with a representation of 5-HT 3 R features and indicate relevant residues, contacts, and types of interactions. Additionally, we expanded the figure content to include WebLogo3 protein sequence logos to indicate sequence conservations, as this is relevant for the type of scientific discussion we wanted to anchor on this image.

Example 2. The gating mechanism scheme of 5-HT 3 receptor

Our first example centered on the small-molecule ligand serotonin, so in this second example, we'll focus on the macromolecule 5-HT 3 R and its gating mechanism. What we will illustrate here is how to create a diagram that summarizes the underlying mechanistic principles of 5-HT 3 R.

In this example, we used the 5-HT 3 R coordinate file 4pir, which includes five receptor chains (A–E), and five chains (F–J) that belong to nanobodies used to stabilize and crystallize 5-HT 3 R. The nanobody components need to be removed prior to any analysis, which PyMOL allows you to do by simply selecting the chains and removing all the atoms associated with them. Because we wanted to display the inside view of the receptor, we also removed three protomers (C–E).

With this clean coordinate file in hand, we created different 5-HT 3 R 3D components in PyMOL. We first rendered the surface of two subunits of the 5-HT 3 receptor using scripts that we are happy to provide to anyone who is interested. Next, we created stick representation for three key residues, W154, V264, and L260, and then we prepared the models for water molecules, as well as Na+ and Cl– ions. We used the command "pseudoatom sol" in PyMOL to create a dummy atom sphere that will be exported from PyMOL and easily duplicated to represent H 2 O, Na+, and Cl– in Inkscape.

Armed with 3D models of serotonin, solvent molecules, and 5-HT 3 R, we moved into Inkscape to assemble the components and optimize the figure elements and layout. We used different drawing tools in Inkscape to represent the lipid bilayer and move it to the approximate transmembrane position of 5-HT 3 R. We also used Inkscape drawing tools to indicate signaling pathway direction; the change of W156, V264, and L260 rotamers; and the domain titling and twisting movements. Our final image captures subtle details of the mechanism and conformational changes, as well as some major points regarding 5-HT 3 R, how it fits within the lipid bilayer, and how it engages serotonin.

These examples show that generating high-quality structural biology images does not require expensive software, fancy hardware, or a great deal of time. We hope that they will inspire you to play around with different tools for structural biology data visualization and develop your own sense of style that represents scientific information with both accuracy and beauty. For those interested in what the actual step-by-step process looks like, we created companion movies that can be accessed via YouTube (here, here, and here).