While microscopes have been used to view countless wonders of the biological world, any object that is shorter than a wave of light, such as an atom, is physically impossible to see with light. For decades, those interested in protein structure have solved this issue by growing protein crystals and shooting x-rays at them. This technique, known as x-ray crystallography, requires multiple synthetic manipulations – these step make the technique less than ideal for discovering how things work in the natural world. A new science fiction-esque technique instead pulses high frequency x-ray lasers at a tiny jet of suspended proteins to observe their structure. This new technique allows for a more authentic interpretation of protein dynamics while overcoming many disadvantages of traditional crystallography.
One of the more anachronistic techniques used in current molecular biology and medicine is x-ray crystallography. Invented by physicists in the early 1900s to solve the structure of minerals, it was applied to structural biology in the 1950s to analyze common proteins such as hemoglobin (the protein that makes blood red) and insulin. Since then, thousands of protein structures have been discovered, leading to invaluable advances in designing drugs and research.
While the last six decades have witnessed many improvements in the equipment used for x-ray crystallography, no fundamental changes in the underlying method have been made since it won the Nobel prize for physics in 1914.
What is x-ray crystallography and why hasn’t it evolved since it was discovered? X-ray crystallography, in short, solves an optical problem. The first lesson of elementary optics is that objects can only be detected using electromagnetic waves that are shorter than the object. It works a bit like a painting – you can’t use a paintbrush larger than the canvas unless your name is Ad Reinhardt. For a quick introduction why, check out our primer on optics and waves.
Essentially, x-rays cannot be focused like normal light, therefore scientists have to use a proxy to determine what a protein looks like. To do this, large crystals of a protein are grown and a single crystal is selected for quality. Next, the crystal is saturated with x-rays and the diffracted x-ray pattern is collected behind the protein crystals. This is back-calculated into a 3-dimensional structure through a complex computational process.
Anyone familiar with x-rays or proteins will realize two issues with this process. The first is that x-rays tend to harm living tissue – saturating protein crystals an x-ray beam destroys the sample. While performing the experiment at cryo-temperatures mitigates x-ray damage, it is still an inherent limitation of the technique. The second issue is that the proteins are crystallized – it’s not often that your meat comes crystallized when you order it at a restaurant. In fact, proteins are never found crystallized in nature. This makes the method both unnatural and difficult from the start.
Enter the Femto-laser. By using Stanford’s Linac Coherent Light Source, super-short bursts of radiation from a particle accelerator capture diffraction patterns from millions of tiny protein crystals. Each burst destroys individual proteins, but not before data is collected. By integrating data from millions of bursts, the protein’s shape is reconstructed. This yields two advantages over traditional crystallography: first, the proteins can be captured in smaller, more “natural” states. Second, the data collected is not limited by the decay of one individual crystal.
In an exciting step forward, scientists have captured a glimpse of how proteins work using the Fempto-laser. Kupitz and colleagues have determined the biophysics of a plant photosystem protein complex. The photosystem is used in the first step of photosynthesis by plants, and is a gigantic complex of proteins responsible for converting sunlight to energy. By exciting the photosystem during bursts of the x-ray laser, they were able to capture two different structures of the protein, showing exactly how it works to convert light to energy. This may take engineers one step closer to generating organic solar panels with much greater efficiency than current photovoltaic or thin-film technology.
This experiment opens the window to a new field of protein chemistry. By using a technique that can monitor dynamic changes in protein structure, scientists may be able to design news drugs for patients or make new synthetic enzymes for industry. While the Fempto laser opens the door to new discoveries, a better technique still waits to be discovered that can observe a protein’s structure in its natural state.
Image: Electron density map of the photosystem complex from Kupitz and colleagues. (Image Credit: link)