X-ray diffraction has been used in the medical and pharmaceutical industry for decades. Given the vast amount of research done in these fields, it is only natural for them to change and evolve, requiring methods of investigation to evolve alongside them. So when the COVID-19 pandemic began and scientists around the world scrambled to learn about it as quickly as possible, x-ray diffraction was one of several highly developed methods available to play a large role in their research.
We all remember hearing the news of a series of distinct pneumonia infections emerging in Wuhan, China in December 2019; word quickly spread globally, along with this novel coronavirus that seemed similar to the severe acute respiratory syndrome (SARS) virus observed in a 2003 outbreak. After the initial emergence of COVID-19, caused by the SARS-CoV-2 virus, the genome had already been shared online by early January. By March, a paper had been published describing the structure of the spike protein on the outer surface of the virus. Important new developments would continue to be made in the coming months, and all of these breakthroughs were possible because of both incredible scientists and sophisticated equipment and techniques for protein analysis.
One of the prominent methods of protein characterization is x-ray crystallography, which uses x-ray diffraction to provide information about the atomic structure of a crystal. When x-rays infringe upon a single-crystal sample, the resulting diffraction pattern can tell researchers the arrangement of atoms within the crystal. In order for this method to work on proteins, a sufficient quantity must be synthesized and then crystallized in a highly ordered lattice. Additionally, the proteins must be robust enough to undergo exposure to x-rays, or instruments must be fast enough to prevent damage to the fragile proteins from the intense radiation during x-ray diffraction.
Key research on the coronavirus protein was performed at several beamlines, including the BESSY II synchrotron in Berlin. With the cooperation of the facility and prior knowledge of the protein due to its similarity to the Middle East respiratory syndrome (MERS) virus that became prevalent in 2012, scientists were able to characterize the coronavirus protease with unprecedented speed. Proteases are enzymes involved in the breakdown of peptide bonds within proteins. With scientists’ newfound information about the protein’s structure, they could start brainstorming potential drug candidates.
Pharmaceuticals often make use of small molecules, which are compounds that alter the activity of proteins when they bind to macromolecules. Structural biologists have been able to use crystallography to observe the behaviour of small-molecule drug candidates with the target protein responsible for the COVID-19 response to see whether, and how, the small molecules bind. When crystallography was used to identify possible inhibitors for the SARS-CoV-2 protease, researchers discovered 66 small molecules that were able to bind to the protein’s active site. Although crystallization is not possible for all proteins, x-ray crystallography was an important step in investigating pharmaceuticals that could treat COVID-19, and many candidates were identified in only a couple weeks of synchrotron experiments.
A new beamline was used by many researchers throughout the US to investigate the components of SARS-CoV-2: Beamline 12-1 at the Stanford Synchrotron Radiation Lightsource. This beamline demonstrates some of the best aspects of x-ray crystallography and points to how x-ray technology can continue to be used in a post-COVID world. For instance, experiments can be done remotely with sophisticated systems in place for data processing, allowing scientists to acquire data without leaving their home laboratories. In addition, the beamline is equipped with a bright microfocus x-ray source, meaning that researchers do not need to produce as much sample to be imaged, and their results come at a higher resolution. Robotics are also integrated into the beamline for remote sample swapping, and researchers could evaluate the proteins within a series of small crystals in rapid succession without the need for growing large crystals. Because of the smaller sample sizes needed, experiments could be completed in a time-efficient manner – and time was certainly of the essence in the initial research being done on the SARS-CoV-2 virus.
The beamline is capable of imaging crystals at room temperature, enabling its use in an early investigation of enzymes associated with replication of the COVID virus with experimental conditions close to body temperature. Another study aimed to elucidate the molecular structures of antibodies that could fight SARS-CoV-2. Advancing x-ray technology such as Beamline 12-1 made it possible to gather important data at an unprecedented speed, even with unprecedented challenges in place due to the pandemic.
While the challenges faced globally throughout the pandemic have been many, the scientific innovations have been numerous and impressive. Vaccines were produced at a remarkable pace, which actually became a pain point for many who felt that the COVID-19 vaccines were rushed. However, starting all the way back in 2008, scientists were using x-ray crystallography to study another messenger-RNA-containing virus called respiratory syncytial virus (RSV). Although the virus is different than SARS-CoV-2, the two have a physical similarity that became a valuable clue to the first COVID-19 vaccine developed years later. Scientists focused on the “prefusion” state of the RSV virus – the structure of the protein before it enters a host cell – and learned its appearance for the first time. Once the imaging was complete, the scientists were able to bioengineer it to prevent it from shifting between the pre- and postfusion states, thus stabilizing the protein. The result was an antigen – a molecule that triggers an immune response – that neutralized RSV 50 times more effectively than any of the previous attempts.
Many other technological advances and instruments were used in the development of COVID-19 vaccines. For example, cryo-electron microscopy, a technique in which 2D images of a protein are generated via low-energy electron radiation, is ideal for proteins that cannot be crystalized. However, x-ray crystallography helped solve preliminary clues about the SARS-CoV-2 structure, potential treatments, and possible vaccines. Its speed, accuracy, and ability to be done remotely were highly important during the COVID-19 pandemic – and as x-ray technology continues advancing, faster, more sophisticated drug discovery will be possible.