In the midst of this pandemic, almost everyone is acquainted with the token Covid-19 protein image. This image has surfaced on all facets of the internet, used as the primary visual in multitudes of articles and media blasts. However, given the extremely small size of the protein, how were scientists able to construct this 3D representation?
Scientists utilized Cryo-Electron Microscopy (Cryo-EM), an imaging technique which involves shooting electron beams at biomolecules to create microscopic snapshots of the individual molecules, which are later used to produce the 3D image of the structure. The breakthrough of utilizing electron beams rather than light beams was due to resolution and wavelength. Theoretically, a much higher resolution image can be produced using electron beams as the electrons wavelength is much shorter (than light) and can uncover sharper and more detailed images than other imaging techniques (Ex: super-resolution light microscopy). Standard Transmission electron microscopes (TEMs) project a beam of electrons through a narrow sample, which interacts with the molecules. The microscope then projects a picture of the sample onto a detector. However, there are many challenges in the TEM technique. Many biomolecules are not well suited for strong beams of electrons and high-vacuum practices involved in standard TEM’s. This process is not suitable for structures made up of organic molecules and biomolecules, as propelling electron beams at living matter can be calamitous. These electron beams cause radiation damage, which destroy the structure. (the beams penetrate the specimen, breaking bonds, and altering structure into burned products-the water abutting the sample molecules evaporates, thus burning and tarnishing the molecules). This explains why in early attempts of electron microscopy, the samples were demolished.
Given the drawbacks of traditional TEM, what other imaging methods were used to capture and study biomolecules in the past?
For decades, researchers have utilized X Ray Crystallography and Nuclear Magnetic Resonance Spectroscopy for imaging. X-ray crystallography involves crystallizing biomolecules and striking them repeatedly with X-rays. Later using the resulting patterns of light [diffracted] to recreate the structure.
This method is able to create superlative structures, but also has certain disadvantages. For the process to work, the biomolecule sample must crystallize, which is not feasible for many biomolecules Many of the body’s biomolecules floppy nature makes them arduous (or even impossible) to crystallize. Even the few that can often require months and years to crystallize. NMR also works best for smaller biomolecules with low molecular weights, and therefore cannot be used to observe much larger proteins or viruses.
Cryo-EM avoids these issues without reducing the quality of the image.
Cryo-EM researchers began working on crystals of the protein bacteriorhodopsin at room temperature. However, by cooling the biomolecules to liquid nitrogen, they were able to derive 4-5x as much data from the images before any substantial radiation damage. While cooling a specimen does not halt radiation damage, but it reduces the consequences of radiation damage 4-5x.
What is the process of Cryo-Em Specifically?
A protein is plunged in a very thin film of liquid (an aqueous solution) and then frozen in liquid ethane (The samples are lowered to cryogenic temperatures). An electron gun then bombards the sample with electrons, at the speed of light, which penetrate through it. An advanced camera then captures 2D images of the sample from different orientations. Finally, the system reconstitutes the different angled images and develops a detailed 3D structure.
The true power of Cryo-EM is the ability to freeze and image a myriad of organic structures: pieces of tissue, solutions of molecules, ribosomes, suspensions of viruses. An example is the freezing of a suspension of ribosomes from a variety of cells. A thin layer of 15-20,000 ribosomes is frozen. As electron beams pass through, images from different angles are taken and then reconstituted to form an average 3d model.
What is the impact?
These images are useful for observing protein function, and malfunction when riddled with disease. Knowing where all the crevices and pockets there are in a protein are vital as they help chemists design drugs which fit into them.
Through this process, scientists using Cryo-EM were able to image and create the famed Covid-19 protein image we see today. By identifying the crevices and the shape of the virus, chemists continue sampling potential drugs to achieve the right fit. This image was the turning point of research, and successfully directed scientists a path to finding the cure.
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Callaway, Ewen. “Revolutionary Cryo-EM Is Taking over Structural Biology.” Nature News, Nature Publishing Group, 10 Feb. 2020, http://www.nature.com/articles/d41586-020-00341-9.
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