Look around! Can you imagine all the pathogens around you? Nowadays, bacteria can be observed using powerful microscopes. But how is it possible to visualize even smaller microbes like viruses?
At the beginning of the twentieth century, it was discovered that waves, called X-rays, could be diffracted by crystals of simple salts. This discovery opened doors that would allow us to study the structural organization of atoms using a technique called X-ray crystallography. In the field of virology, this technique was first used for the determination of the structure of the Tobacco Mosaic Virus in the 1940s, followed by the determination of the icosahedral symmetry of the Tomato Bushy Stunt Virus. To commemorate the importance of X-ray crystallography in understanding biology, the International Year of Crystallography was celebrated in 2014.[If you want to learn more about the most relevant milestones in crystallography, click here]
To obtain the three-dimensional structure of a biological sample, it needs to be crystallized in a single crystal that is then placed in an X-ray machine, where it is bombarded with X-rays. The crystal diffracts the X-rays, and the pattern of diffraction is recorded by a detector in the form of spots. The three-dimensional structure is then calculated using complicated mathematical operations.[If you are new to crystallography, we recommend this movie]
In some cases, it is not possible to use X-ray crystallography to determine the three-dimensional structure, because some samples are just too difficult to crystallize. In these cases, the alternative is to use cryo-electron microscopy (cryo-EM). With this method, instead of crystallizing the sample, you just freeze it. However, cryo-EM can still be limited in terms of resolution when compared to X-ray crystallography.
To obtain the three-dimensional structure using cryo-EM, the frozen sample is placed under a microscope, where instead of using light like in a regular microscope, a beam of very tiny, negatively charged particles, called electrons, are used. These electrons are then scattered and recorded from different angles. To understand it better, imagine that you are in front of a field with several cows and you want to determine the structure of a cow. In this case, several pictures of the field with cows is taken from different angles. Then, you choose a few cows from one picture, and try to find these same cows in all the other pictures. Using these pictures and elaborate mathematical operations, the three-dimensional structure is determined.
In short, X-ray crystallography requires the production of a crystal, but provides accurate information at the atomic level! On the other hand, cryo-EM overcomes these limitations but is limited by low contrast and prone to artifacts caused by sample freezing and heterogeneity. Thus, both techniques complement each other in determining structural information on viruses.
Knowing the structure of a target protein provides insights into its function. Current techniques like X-ray crystallography or cryo-EM can be used to learn more about the structures of viruses and viral components. These structural studies often serve as a basis for the rational design and development of antiviral drugs. One good example of an antiviral drug developed based on the knowledge of the structure of the virus is the drug Tamiflu® (generic name Oseltamivir), especially well-known after the swine flu pandemic in 2009. This drug was specifically designed to inhibit a distinct part of the influenza virus using structure-based drug design.