Two beams are better than one

As neutron crystallography advances it is becoming a powerful scientific tool, but will it replace its well-known X-ray cousin? Not so say Matthew Blakeley and Derek Logan – rather, they will make a most formidable partnership… 

More and more frequently, scientific facilities that use a variety of analytical tools are coming together to solve challenges within the scientific community.

Though there are relatively few locations around the globe that house the most powerful instruments, such as neutron and X-ray beams, their impact on the scientific community is great. Almost all areas of research have the potential to make progress with the help of these potent analytical weapons.

X-rays are likely one of the first techniques that come to mind when we think about making the invisible visible. Many of us will have undergone or experienced X-ray scans of a broken bone, which hides beneath many layers of skin and muscle. Yet, a clear, crisp image is produced for analysis. In these cases, X-rays are passed through the body during the scan, and certain structures, such as bone, absorb the radiation at different rates. This allows the different tissue types to be distinguished, and the fractures or abnormalities to be identified.

Outside of hospitals, X-ray radiation is increasingly useful in laboratory environments, in the form of X-ray crystallography. Here, a far more intense beam of X-rays is aimed at a sample, and interacts with the electrons in the material. This produces a detailed image of the atomic structure, and helps scientists to understand the properties of the material and predict its behavior.

A perhaps lesser known, but equally powerful technique for investigating materials is the complementary neutron crystallography. Intense beams of neutrons, when fired at a sample, are able to explore deep inside matter via interaction with the nuclei. This provides an incredibly detailed picture of the atomic structure, and additionally of the magnetic interactions within the material, which X-rays cannot provide.

Combined expertise

These specialist techniques, which can be carried out at only a few facilities worldwide, have often come together to provide researchers with a detailed and complete picture of what is occurring deep inside the structures they work with. Here at the Institut Laue-Langevin (ILL), the world’s flagship neutron science facility, we share a site with the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Our two institutions hold the world-leading instruments in neutron and X-ray crystallography respectively, and have offered up our combined expertise and equipment in a number of research collaborations.

As ILL’s neutrons are non-destructive and can explore deep inside matter, they are an ideal probe for most materials, including biological or sensitive materials that may be perturbed by X-ray radiation. They interact with nuclei and magnetic moments of unpaired electrons, while ESRF’s high-energy X-rays interact with most electrons in a material, making them outstanding complementary techniques. Neutrons are perfect for determining the position of light elements within a substance, such hydrogen or lithium. They also have an exceptionally powerful penetration ability, despite their low energy. X-ray beams at modern synchrotrons, an extremely powerful X-ray source, also achieve incredibly high-resolution images. Thus, the combined methods allow us to study the structure of a variety of materials with unparalleled attention to detail.

A recent study¹ we carried out at the ILL exemplifies the powerful partnership of neutrons and X-rays in health-related research. In a collaboration with Lund University (Sweden), Oak Ridge National Laboratory (USA) and Heinz Maier-Leibnitz Zentrum (Germany), we investigated a sugar-binding protein, galectin-3 (galectin-3C), which is associated with breast cancer and other chronic conditions including heart disease. For the first time, using the LADI-III beamline at the ILL, we were able to identify the binding characteristics of the protein. This will be key in understanding how to optimise future drug design to tackle the related diseases.

In normal bodily function, galectin-3 is involved in lots of physiological processes in the body, notably in pathways involving cell-to-cell adhesion. This involvement may be associated with the increased expression of galectin-3 shown in a variety of tumours, as it can enhance the adherence of cancerous cells to other cells in the body, including the extracellular matrix that connects many tissues and organs. This suggests it may also have a role in metastasis, the process by which cancer spreads throughout the body².

Until now, our understanding of the exact mechanism by which the protein binds has been mostly guesswork. X-ray crystallography has allowed researchers to build a very detailed picture of the structures involved, and enabled the visualisation of all of the heavier atoms in the structures. This was key in mapping the structure of the protein in a ligand-free state, before any binding begins. The next step in the characterisation requires the exact location of the hydrogen atoms in the binding process, which is where neutron crystallography is the ideal tool. Neutrons can pinpoint these lighter atoms, whereas X-rays alone can only infer the exact locations using sometimes-unreliable hypotheses based on physics and chemistry.

By understanding the positioning of the hydrogen bonds through neutron crystallography, we have paved the way for better drug design that targets how galectin-3 attaches to other proteins in potentially disease-causing pathways. An accurate picture of how this protein might facilitate the spread or binding of tumorous cells will better serve the development of cancer pharmaceuticals. In the UK, someone is diagnosed with cancer every 2 minutes³, and breast cancer is the most common of them all, accounting for 15% of all new cancer cases in 2015⁴.

Hydrogen bonds

As we demonstrated, neutrons are particularly useful studying the molecular make-up of biological materials. However, X-ray crystallography has a much longer history of successfully characterising biological structures, being highly influential in understanding the structure of DNA and proteins for the first time. Its reliability and penetrating power makes it the go-to method for solving the structure of large biological molecules. Nevertheless, neutrons have earned their place amongst the world’s most powerful probes thanks to more recent advances, allowing instruments to look at smaller crystals and collect data faster. The LADI-III beamline instrument is a particularly formidable probe of biological samples, due to its ability to locate and visualise particles of interest even amongst water molecules, which are particularly mobile and difficult to find.

This tool was very influential in a study at the ILL⁵ that also characterised a binding process: in this case, the binding of anti-retroviral HIV drug (amprenavir) to its target enzyme, HIV-1 protease. Currently, there are approximately 36.9 million people living with HIV and tens of millions of people have perished from AIDS-related causes since the start of the epidemic in the 1980s⁶. HIV not only affects the health of individuals, it affects households, communities, and the development and economic growth of nations – there is still no cure.

In the collaboration between the ILL, Georgia State University, and Oak Ridge National Laboratory, X-ray crystallography was used to gain information about the mechanism, and researchers were able to identify that there were likely several important hydrogen bonds in the process. However, X-rays beams are unable to pinpoint hydrogen atoms accurately. This is where neutrons came in to finish the job. Neutron crystallography showed that there were only two, strong hydrogen bonds existing between the drug and the enzyme. This demonstrates new targets for improvement by pharmaceutical designers, and suggests the effectiveness of the drug can yet be improved.

Material advancement

Outside of making an impact in the pharmaceutical industry, the combination of neutrons and X-rays are helping with the development of advanced materials in a number of fields, including aerospace. At the ILL along with our neighbours, the ESRF X-ray facility, we recently announced an agreement⁷ with two prominent European aerospace companies to identify and tackle material challenges in the industry. OHB System AG are satellite producers, creating products for applications from telecommunications to space exploration. Its sister company, MT Aerospace AG, is a technological leader in the production of lightweight metal and engineering for applications in space vehicles and aeronautics. The aim of the arrangement is to bring together the practical and intellectual capabilities of the research facilities to further the field of aerospace materials.

Despite the substantial opportunities that come with such a collaboration, the partnership of neutron and X-ray facilities has largely been limited to academic institutions. This is set to change, thanks to the help of a dedicated group of engineers and scientists from the ILL and ESRF, who will help identify materials that are struggling in the hostile environment of space by examining the atomic structure of the materials. The understanding of the materials provided by the deep-penetrating techniques of ESRF and ILL makes the development process of new technologies faster and easier, wasting less time, money, and resources.

Neutron and X-ray investigation allows current and prototype materials and processes to be scrutinised with immense detail, providing a toolkit to adjudicate them over all relevant length scales. It should allow companies to make lighter and more durable materials for the harsh surroundings of space exploration, and yield technologies of the utmost possible consistency and efficiency. This is likely to secure their place ahead of competitors. Neutron and X-ray probing can enable this progress specifically as it allows the organisations to examine specific components of their products such as valves or tank structures in rockets, and electronic components and circuit boards in satellites, where avoidance of destructive techniques is essential.

This arrangement demonstrates that not only are neutrons and X-rays a formidable partnership when it comes to extricating the details of structures, the facilities can also provide industry with exclusive and confidential access to instrumentation that will keep them on top of the market.

X-ray crystallography has provided scientists for decades with an incredible level of detail when it comes to understanding any materials, from the organic to the metallic. While advances in neutron science have certainly resulted in many advantages that cannot be achieved by X-rays, it is unlikely that the latter will ever become obsolete.

Neutrons generally require significantly larger crystals for examination than X-rays, which has been the limiting factor in past years. The opportunities provided by the two techniques are best appreciated when they are combined in a potent analytical team.

References

1. https://www.ncbi.nlm.nih.gov/pubmed/29672051
2. https://www.sciencedirect.com/science/article/pii/S0304416505004083
3. https://www.cancerresearchuk.org/health-professional/cancer-statistics/incidence#heading-One
4. https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/breast-cancer#heading-Zero
5. https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201509989
6. UNAIDS. How AIDS Changed Everything; 2015.
7. https://www.ill.eu/fr/infos-presse-evenements/press-corner/presse-et-infos/big-science-and-industry-join-forces-to-innovate-new-space-technologies/

Authors:

Matthew Blakeley is LADI-III beamline scientist at Institut Laue-Langevin (ILL) 

Derek Logan is Associate Professor in structural biology at Lund University