A Beginner's Guideline for Low-Cost 3D Printing of Macromolecules Usable for Teaching and Demonstration

The structure and function of biomolecules relationship is the hallmark of biochemistry, molecular biology, and life sciences in general. Physical models of macromolecules give students the possibility to manipulate these structures in three dimensions, developing a sense of spatiality and a better understanding of key aspects such as atom size and shape, bond lengths and symmetry. Several molecular model systems were developed specifically to represent particular classes or groups of molecules and hence lack the flexibility of a universal solution. Three‐dimensional printing could nevertheless provide such a universal solution, as it can be used to create physical models of biomolecular structures based on the teacher's or demonstrator's needs and requirements. Here, insulin was used as a model molecule and several depiction and printing parameters were tested in order to highlight the technical limitations of the approach. In the end, a set of settings that worked is provided which could serve as a starting point for anyone wishing to print his or her own custom macromolecular model on the cheap.

Keywords: 3D printing; general public; insulin; molecular models

From pdb file to physical models of macromolecules using 3D printing.

One of the most important concepts in biochemistry, molecular biology, and life sciences in general is the connection between the structure of biological molecules and their function.1 Students usually first encounter molecular structures in the chemistry class as two‐dimensional (2D) structural formulae.2 Although highly useful and convenient, these representations are often received by biology and medical students as simple drawings. Important aspects such as atom size and shape, bond lengths and symmetry, and generally a sense of spatiality and dimensionality are lost. Due to the complexity of proteins and nucleic acids, this is especially true for these macromolecular structures of great biological importance.

Physical models of macromolecules give students the possibility to manipulate these structures in three dimensions and have been shown to be preferred.3–5 These physical three‐dimensional (3D) representations allow students to better overcome the problems associated with the translation between the 2D formulae into the 3D space.6 For this reason, numerous models have been developed that are using folded paper, polystyrene, glass, or plastic beads.7 Molecular model kits such as Molymod8 containing plastic spheres and connecting rods are commercially available. A really nice and growing collection of folded paper models for proteins and nucleic acids is readily available, for example, on PDB‐101, the educational portal of RCSB‐PDB.9

However, the majority of available models are developed specifically to represent a particular class or group of molecules. They lack flexibility and fail to provide a universal solution capable of representing a large variety of chemical and biological structures.7 A Molymod teacher kit, for example, contains at most 300 atoms and 150 links, which is not enough to create the 3D structure of a small protein such as insulin. Also, there is no way of representing insulin using a Molymod kit as it is most commonly depicted by molecular modeling software: cartoon and surface.

These limitations of traditional molecular models can be solved by using 3D printing to create physical models of biomolecular structures based on the teacher's or demonstrator's needs and requirements. 3D printing is an additive manufacturing process in which object is built from a series of fused cross‐sectional layers. The layers of material are fused in succession under computer control to make a three‐dimensional physical object from a digital model. With 3D printing, molecules can be created in one piece directly from a digital 3D design file, greatly simplifying the process of fabricating molecular models.7

Although technically difficult and expensive at its debut, the 3D printing field has nowadays reached a level of technological maturations and standardization that makes it easily applicable in a teaching environment. Moreover, there are a variety of different 3D printing technologies and materials to choose from. For example, some 3D printers deposit thermoplastics or ultraviolet resins for the individual layers, whereas other 3D printers fuse composite powder particles together through the use of liquid binders or lasers.7 Fused deposition modeling (FDM), also referred as fused filament fabrication is a 3D printing technology that relies on melting and extruding a thermoplastic filament layer by layer that is by far the cheapest and most readily available. FDM printers can be found fewer than 500 euros, while 1 kg of thermoplastic filament such as polylactic acid (PLA) is priced at around 20–30 euros. The low cost and the ability to design and fabricate your own custom object make FDM printing highly attractive for creating physical models of macromolecules in labs and universities, or even high schools from low‐income countries. The models can be used for teaching at large classes or can be given to students to interact in groups or individually, helping them to better understand key concepts in life sciences.

There are several published works that describe the process of designating and fabricating a physical model of a molecule using 3D printing. The most notable are References 1,2,10,11, while the excellent work of Da Veiga Beltrame et al.12 is very detailed and focused on printing proteins and nucleic acids models. The NIH 3D Print Exchange server (https://3dprint.nih.gov/) offers a fully automated pipeline to convert coordinates of macromolecular structures into printable files. Nevertheless, all assume that the user has some technical background or experience on 3D printing which, most of the time, is not the case for a biologist. A first‐time user of a 3D printer will find itself in the position of tackling many decisions regarding the generation and printing of his desired molecular model. The current guideline is based on and will go hand in hand with the instructions provided Da Veiga Beltrame et al.12 and aims to provide a guideline on the dimensions and settings to be used to generate usable models for proteins and nucleic acids visualized as molecular surface, balls and sticks and cartoon, or any combination of the above. Our main philosophy and focus is on making these models as cheap as possible, thereby only open source or free for academic software was used to generate and prepare the models for printing. All models were printed on a stock, under 500‐euro FDM printer. We aim to provide a set of settings that worked and could serve as a starting point for anyone wishing to print its own custom macromolecular model on the cheap. The data presented here are focused on printing on the smallest scale in order to highlight the technical limitations of the approach.

Pig insulin (PDBID 4INS13) was used as a model molecule for testing different model generation and printing parameters. The asymmetric unit of 4INS has many key features of larger proteins14: it contains the most important secondary structures, coordinates metals, and has a quaternary structure. Its small size is a major advantage in this case as it translates into much faster printing times. The 3D coordinates were downloaded from RCSB PDB as a.pdb file and used to generate different models.

The general workflow for creating a macromolecule model to be printed using a FDM printer is based on the work of Da Veiga Beltrame et al.12 and is graphically depicted in Figure 1. UCSF Chimera v.1.1415 was used to set‐up the model (visualization mode, features to be highlighted, depiction parameters) and to convert it to an stereolithography.stl file. The resulting.stl file was fixed using the Automatic Part Repair function in Autodesk Netfabb Premium 2020 in order to improve its printability and exported again as a.stl file. The model was oriented and sliced for printing using Ultimaker Chimera 4.6.1 with the Auto‐Orientation plugin installed.

A Creality Ender‐5 Pro with automatic bed leveling and a single extruder was used for printing using the following configuration, unless stated otherwise: 0.4‐mm nozzle, layer height 0.2 mm, 80 mm/s print speed, 200 mm/s travel speed, 4 mm retraction, 45 mm/s retraction speed, support was automatically generated everywhere, support overhang angle 55°, support density 20%, concentric support pattern, 8‐mm brim for adhesion. All models were printed on generic PLA (FormWerk, Bucharest, Romania) at 190°C with the bed heated at 60°C. After printing the support material was mechanically removed with a set of pliers. The physical dimensions of the printed model were measured using a Vernier caliper.

FDM printing works by depositing layer over layer of thermoplastics. The consecutive layers adhere to each other upon cooling and create the solid 3D object. Each new deposited layer needs to be supported by the layer beneath it. For this reason, the object to be printed on an FDM printer is generally designed and oriented in such way that there are minimum overhangs or steep angles. Macromolecular structures on the other hand are abundant with pockets and protuberances or with bonds, atoms, and other parts that start on thin air. The solution for this problem in FDM printing is the introduction of support material. In the step just before printing, the software responsible for preparing instructions and controlling the printer (termed slicing software, Ultimakes Cura in our case) automatically designs some support structures that start from the build plate and allow for the overhanging structures to be successfully printed on. This adds time and material cost to any print and the extra material is sometimes difficult to remove but is mandatory for any complicated model such as biological molecules. An elegant solution is to print the support structures using a water soluble plastic (e.g., polyvinyl acetate), while the model itself using insoluble PLA. After printing, the support material is removed by simply submerging the model in water. This approach nevertheless requires a 3D printer equipped with two extruders and hot‐ends, which adds to the general costs. Printing with a single material is easier from a technical point of view but requires extensive postprocessing to mechanically remove the support structures.

The structure of a macromolecule can be depicted and printed in various ways. Ribbons or cartoons models are good for highlighting the structural motifs such as helices and sheets. In balls and sticks models, all the atoms are shown as small spheres connected by rods corresponding to the chemical bonds. When visualized as surface, it is the surface that another molecule (like water) would see. This surface is obtained by probing the spheres representation using a probe of arbitrary radius.15 These modes of visualization can be easily combined in Chimera depending on one's need. For example, cartoons can be used to depict the secondary structure of a protein, while the catalytic amino acids side chains and substrates can be represented as balls and sticks on the same model to argue their position and interactions.

Each of these representations requires different settings when printing in order to get a physically sturdy and solid molecular model as economically as possible. Moreover, these printing settings also depend on the printing scale or the final dimensions of the printed object. For this reason, the one of the first steps when printing a macromolecule structure is deciding on the size of the physical model. Chimera generates models at a consistent scale, at 107 magnification, disregarding the level of zoom applied when visualizing a molecule. In our case, 1 Å in Chimera roughly prints as 2 mm when using 100% scale in Cura slicing software (see Table 1). Depending on the molecular model, printing at high scales might generate large physical models that might not fit the printer's printing volume or that takes too much time and plastic to print. Printing at too low scales leads to low details, fragile models, or support material that is impossible to remove.

1 TABLEChimera depiction parameters and dimensions of the printed model when scaled at 100%

1 Abbreviation: N.D., not determined.

2 a Pseudobond radius was same as height.

This representation is great for arguing the complementary of molecular shapes and is by far the easiest to print. Following the instructions provided by Da Veiga Beltrame et al.,12 including leads to good quality models. Chimera‐generated models can be printed at scales as low as 50%, anything lower than that and the atomic details are no longer visible (Figure 2). The support material is easy to remove, unless is buried within a pocket.

Surface models are generally strong and robust and can be printed with only two layers for the wall (wall thickness 0.8 mm). The insulin model was successfully printed at 100% scale with just one wall layer and no infill, but the printed object was fragile and broke down during the removal of support material. No infill was required at scales of up to 200%. Over this scale, an infill of 5% is required to support the bottom wall of the cavities.

This representation is great for explaining the secondary and tertiary structures. It can also be used as a simplified representation of nucleic acids. The default depiction parameters in Chimera do not generate printable models. The thickness of key components of a model (helices, sheet, coils, ladder, and rung) needs to be increased and additional bonds needs to be added to for a better rigidity and sturdiness of the model. Da Veiga Beltrame et al.12 do a great job in explaining how this should be done but indicate only one set of dimensions. While the set is generally good, it does not work when one needs to print large molecular models at a scale below 200%.

Taking insulin as a model, we generated a set of printable.stl files in Chimera with the depiction parameters indicated in Table 1. All models were printed at 100% with two wall layers, the support material was removed and the quality of the models as well as the dimensions were assessed. Some models failed to print, some broke while postprocessing (Table 1), indicating that the minimum diameter for a coil regions and bonds is at least 2.6 mm (or 1.45 Å in Chimera when printing at 100% scale).

The final dimension of any element in a printed model can be easily calculated using the following formula:

$$\text{Printed dimensions}\left(\mathrm{mm}\right)=2\times \text{Chimera dimensions}\left(\mathrm{\AA }\right)\times \text{Cura Scale factor}\left(\%\right)/100.$$

Models A, B, C, and D that failed to print at 100% were further scaled such that the thinnest elements in the model (coils and pseudobonds) were 2.6 mm and then reprinted. All models printed successfully and are detailed in Figure 3.

The cartoon model of proteins and nucleic acids are generally more fragile than the corresponding surface models. Although printing with two wall layers and 20% infill does allow the production of sturdy models at a scale of 100%, for a higher scale we generally use four layers for the walls. This means that any part that is lower than 3.2 mm in diameter will actually be solid.

Due to the sheer number of atoms and bonds that make the model utterly complicated, the utility of representing whole proteins as balls and sticks is debatable. Moreover, the same complexity requires extensive usage of support material when printing balls and sticks models. Removing the support structures is tedious and sometimes impossible work.

Following the same rule of thumb that the thinnest element in the printed model should be 2.6 mm diameter, we generated several insulin models using balls and sticks that were printed at a scale of 100%, 150%, 200%, and 300%. Although all were printed fine, the support material was impossible to remove without breaking many amino acids side chains from model. A dual extruder printer capable of printing with soluble support material is required for printing such complicated models. For other models, like a protein alpha‐helix, a beta‐sheet, and a DNA dodecamer, we successfully printed balls and sticks models at scales ranging from 350% to 450% (Chimera depiction setting and photos with the printed models are available as Figures S1–S3). A model yeast phenylalanine transfer RNA was also printed at 200%, but the model again broke into several pieces during postprocessing (Figure S4) and had to be glued back together.

This depiction mode is nevertheless great when combined with cartoon or even surface to highlight specific structural features (Figure 4).

Models presented in Supporting Information are currently used by the author for teaching structural biochemistry at undergraduate level. The lectures are taking place at the Biology Department from the author's host institution and are part of the first‐year curriculum. The 3D‐printed models are used after the freshman students have been introduced and have used a molecular model kit such as Molymod. The 3D‐printed models are handed over to students and specific structural features of the models are highlighted based on discussions: position of the side chains and H‐bond formation for the protein secondary structures (models depicted in Figures S1 and S2); base‐stacking, small and large groove, base accessibility for DNA (Figure S3); quaternary structure, size differences, and shape complementarity for hemoglobin/hem (Figure S6). As the students handle the models and the discussions are taking place, the educator also displays the same molecules in PyMol on a video projector and helps them find the right orientation and view angles.

As 2020–2021 is the first academic year when the 3D‐printed models are used and the implementation is still in progress, no outcomes data are available. Generally speaking, the students were intrigued by both the models themselves, and by the technology used to generate them. One downside is that upon their first contact with the models, the students focus was more on the 3D printing technology and not on the significance of the model itself. After the first contact, their focus shifted, and they were able to better pay attention to the discussions tacking place.

The data presented here prove that cheap FDM printers can be used to produce custom physical models of macromolecules useful in an academic environment. When printing a macromolecular model, one needs to strictly correlate the model generation parameters with the depiction mode and the final scale of the model. A good starting point to follow is printing the thinnest elements at a diameter of at least 2.6 mm. Models depicting molecular surface can be printed at a scale of 50% or higher, cartoons models at 100% or higher, while balls and sticks models are difficult to print and require a scale of at least 350%.

The author declares no potential conflict of interest.