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X-Ray Crystallography: Growing Protein Crystals in Microgravity

What is X-ray crystallography?

X-ray crystallography is an experimental technique used to determine 3D molecular structures. In biochemistry, it is a key method for the structural analysis of proteins and biological macromolecules.

Traditionally, light microscopes are used to study small objects. This is effective for whole cells and tissue samples. However, the resolution (i.e. the shortest distance in which an imaging system is able to differentiate between two objects) is limited to objects that are about the same wavelength as light. Therefore, microscopes are not always suitable for imaging much smaller molecules. For this reason, techniques such as X-ray crystallography are employed.

The principle of X-ray crystallography is to convert the X-ray diffraction pattern of your sample into an electron density map representing its 3D structure. Working at the electron level allows for the resolution of incredibly small molecules. Dynamic interactions such as enzyme activity and receptor binding can also be studied through X-ray crystallography.

How is it relevant in terms of scientific research?

Determining 3D structures of biological macromolecules at atomic level resolution through x-ray crystallography can be advantageous to many important fields in biology. Furthermore, the structural information of a molecule also provides insights into its functions and interactions within an organism. Such information can help identify regions to target for drug development such as cell surface receptors. It also allows for the creation of antibiotics specific to bacterial enzymes and the detection of biomarkers for diseases. As the resolution achievable through X-ray crystallography experiments increases, the quality of information we gain from the data improves substantially. It is therefore essential to optimise this experimental process as much as possible.

How is X-ray crystallography carried out?

1) The formation of crystals

The first step in X-ray crystallography is to produce your crystal. A large amount of purified sample is required for crystallisation and there are many methods that can be used.

The main principle of crystallisation is to precipitate the purified molecule very slowly from solution so that it crystallises. This is often carried out by altering the solvent by slowly adding precipitating agents (e.g. ammonium sulfate) until the sample precipitates. Unlike salt crystals, an open evaporation environment is not suitable for biological molecule crystallisation because structures such as proteins fall apart outside of an aqueous environment.

Figure 1: Keywords for the components that make up the crystal growing part of an X-ray crystallography experiment.

A commonly used technique to grow crystals is vapour diffusion. This can be set up as either sitting a drop or hanging drop method. It relies on the concentration difference between the solute in the reservoir and in the droplet. This establishes a concentration gradient that drives the diffusion of water vapour from the droplet to the reservoir. As this happens, the droplet shrinks, and the solute concentration increases, building up the crystal.

Once the sample is slowly precipitated out of solution, monomers will build around the solute front and a crystal containing billions of monomers will form. Crystals amplify X-ray scattering and these diffraction patterns can thus be detected computationally.

2) X-ray diffraction

Once crystals are successfully obtained, they are exposed to an X-ray beam. The X-rays get diffracted through the crystalline structure, producing a specific X-ray diffraction pattern. Each electron in the structure that is hit by an X-ray beam will emit a scattered X-ray, which will be picked up on the detector.

Figure 2: The set-up of crystal and X-ray source to create a diffraction pattern, with the X-ray diffraction recorded by the detector.

If we look at two neighbouring electrons, both of which have scattered X-rays, the scattering pattern will resemble the double slit experiment, creating points of maxima and minima on the detector.

Figure 3: An overlap of scattered X-Rays from two neighbouring electrons. The detector will pick up maxima and minima from the overlapping rays.

In X-ray crystallography, this takes place on a much smaller scale and with all of the electrons in the crystal structure. The resulting diffraction pattern will therefore be very complicated, as it is created by the overlap of many electrons. Although the final diffraction patterns bear no physical resemblance to the molecule that has been crystalised, there is a defined relationship between diffraction patterns and the molecules. We can use this information to determine clear molecular structures.

3) How do we construct the structures from the density map?

We can produce our image of the molecule from the diffraction pattern using Fourier transform, a mathematical transformation able to take a sum of the waves scattered from every point in the crystal and convert them into electron density maps.

Using these maps, amino acids or monomer sequences can be manually built in using interactive computer graphic programs (e.g. Coot). This allows the display of the model in 3D and allows manipulation of the structures to optimise the results (e.g. inserting/ deleting residues or rearranging side chains).

Why are crystals challenging to grow?

The process of growing crystals is the most challenging step of X-ray crystallography, making it the bottleneck in these structural experiments. Good crystals are essential for obtaining good data, which means large, symmetrical proteins with few impurities are ideal.

Protein crystals are fragile and require gentle handling to prevent breakage. This includes picking them up with pipettes immersed in solution. Crystals formed also contain large solvent channels, with the percentage within protein crystals ranging from 30 to 70%. The presence of these solvent channels mimics the native environment of a protein within the crystal. If these channels dry out, it may lead to denaturation of the proteins.

It is also important to note that the solvents themselves may not be very physiologic (e.g. have high pH or salt concentrations), therefore these changes to the proteins must be taken into consideration when interpreting its 3D structure.

What conditions on Earth adds to this challenge?

An ideal environment for good crystal growth is one where the only movement within the solvent is caused by diffusion alone. This ensures slow monomer movement from the solvent to the growing crystal (solvent) front. When a crystal is allowed to grow very slowly, it reduces non-uniformities and defects which impact the resolution of the final density maps.

Due to the gravity on Earth, convective currents are often established in the bulk solution of crystal growth experiments. In gravity, heavier fluids fall and lighter fluids rise, leading to convection. When this occurs, dissolved solutes are transported rapidly through the solution, where they can encounter the solute front in an unorganised and undistributed manner. Amorphous precipitates are frequently formed as a result, which is a non-crystalline solid lacking organisation of molecules in a definite lattice.

In fact, with convection, amorphous precipitates are kinetically favoured thereby preventing crystal formation. With micrometre sizes, any alterations in the crystal structure will have significant effects on the resolution and degree of structural determination. Without gravity, convection currents cannot occur. Therefore the only way to remove these currents is to grow crystals in a microgravity environment.

What is microgravity and how does this environment on the International Space Station solve this issue?

Microgravity is the phenomenon which gives astronauts the appearance of floating in the International Space Station (ISS). In reality, the Earth's gravitational field extends out into space much further than the orbit of the ISS, so these conditions need to be manually created.

Establishing a microgravity environment depends on two things: a vacuum and an object in free fall. Space is an example of a vacuum, which means it is absent of any particles. Thus, when an object falls, there is no air resistance acting on it. Once we remove air resistance, the speed at which an object falls is independent of its mass and is influenced by gravity alone. At the same position in space (in this case in the ISS), all objects experience the same effect of Earth’s gravity and therefore will all fall at the same rate.

The next factor is free fall. To create this on the ISS, the free fall of the shuttle is parallel to the curvature of the Earth; this maintains a constant orbit of the shuttle around the planet. This requires extremely specific speeds and heights of orbit to maintain; the ISS is currently travelling at 7660ms-1 at an orbit height of 408,000m falling freely in vacuum, thereby establishing a microgravity environment.

The microgravity on the ISS solves the issue of convection currents in crystal growth solutions. This means that larger crystals with far more ordered structures are able to be grown on the ISS.

What experiments have already been carried out on the ISS?

The uses of X-ray crystallography extend past structural analysis. In many pharmaceutical companies, monoclonal antibodies (mAbs) are being developed as new drugs. mAbs have had a large impact on treating oncologic, neurological, and cardiovascular diseases and can be engineered to bind to specific substances. This can include cell surface receptors and subcellular targets on both human and pathogen structures. Over 70 therapeutic mAbs are now available for treatment in the United States and across Europe.

Effective treatment via mAbs requires high doses of mAbs and can only be administered intravenously over the course of a few hours. The formation of crystals with a high concentration of mAbs could shorten the time for administration significantly, giving a patient the correct dosage with only one injection. Obtaining mAbs in a stable crystalline form also ensures its preservation at a range of temperatures, which is essential as methods of refrigeration are not accessible to everywhere and would also have a half-life long enough to ship and store worldwide.

Merck Sharp & Dohme Corp (MSD), in association with the ISS National Laboratory, grew crystals of the Pembrolizumab mAb, used in treatments for a range of cancers such as esophageal cancer, over the course of 10 SpaceX missions between 2014 and 2016. They found that in microgravity, much larger crystals were being produced, up to 5 microns in size, without the impurities that often develop on Earth.

Figure 4: Pembrolizumab crystals grown on the ISS in microgravity conditions (left) compared to smaller crystals grown in laboratories on Earth (right). Image courtesy of NASA.

Similarly, the University of Toledo carried out an experiment on SpaceX's 18th commercial resupply services mission in 2018. The species being crystallised here was tryptophan synthase, an enzyme essential for the growth of salmonella and other bacteria and therefore an excellent target for antibiotics. In this experiment, enhanced crystal growth on the ISS compared to on Earth was also observed.

Looking forward - the future of X-ray crystallography

Future experiments have already been planned to be undertaken on the ISS National laboratory. These include determining the structure of the aspartate aminotransferase enzyme in humans which acts as a biomarker for hepatic and cardiovascular diseases and Ribonuclease H protein which is involved in DNA repair and could be exploited in cancer treatments.

As well as new experiments, optimisation of the x-ray crystallography process is being carried out. Hydrogen atoms in biological macromolecules are being replaced with deuterium, an isotope of hydrogen with a neutron as well as a proton. Although deuterium has a higher mass number than hydrogen, its chemical properties are the same. The crystallisation is then carried out with deuterium instead of hydrogen, which is called perdeuteration.

Introducing more neutrons into the protein crystals creates a new type of crystallography called neutron diffraction. It does not require crystals as large as those used in X-ray crystallisation and can generate higher resolution images. Combining this advancement with a microgravity environment could produce crystals of much higher quality than are currently achievable on Earth. Using this for biological and medical research could revolutionise the field of drug development, as well as giving us in-depth understanding of several biological processes.

Some supplementary materials for you

Author: Ambre Bexter, BSC Biochemistry

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