Cryo-electron Microscopy – The Technique Uncovering The Secrets of Structural Biology

It’s a commonly cited fact that we know more about the farthest reaches of space than we do about the deepest depths of the ocean. But perhaps even less is known about the inner workings of cells.

Many of us will remember the diagrams of cell structures we learnt in our basic biology classes, crudely represented by a vaguely circular or rectangular blob. Zoom in a little closer and that blob is actually an intricate network of interacting moving parts – the proteins that comprise the cell’s machinery. But what do those proteins actually look like?

With too many intramolecular interactions to easily predict their folding, and on a scale too small to be easily visualised, scientists are still struggling to determine the structures of complex proteins.

Throughout the last few decades, the visualisation technique cryo-electron microscopy (cryo-EM) has been gaining traction, and has been able to provide some of these answers.

Transmission electron microscopy (“TEM”) has been around for many years, being first demonstrated in 1931 by scientists Max Knoll and Ernst Ruska – who would later go on to win a Nobel prize for their work developing the technique.

In this technique, electrons, in a focused beam, are transmitted through a sample with which they interact before hitting a detector. The sample absorbs some electrons and causes some electrons to scatter, wherein the pattern of scattering is dependent on the density and crystal orientation of the sample. The electrons which have not been absorbed or scattered then hit the detector, and from this “shadow” it is possible to gain an insight into the structure of the sample.

One reason why TEM was initially so exciting for those wanting insights into cell structure was the potential level of resolution it provided. Higher resolution images are produced by imaging with shorter wavelengths. When accelerated to extremely high speeds (as electrons are in TEM), the wavelength of the electrons is very short – thus giving them the potential to deduce details on a nanometre scale.

The issue, however, with this technique was that the use of such high-energy electrons had the potential to cause real radioactive damage to any delicate biological samples viewed. As long ago as the 1960s, it was proposed that lowering the temperature of samples may reduce the amount of damage caused. However, it was not for another 20 years before the TEM-based technique now known as cryo-EM was realised.

Jacques Dubochet and his team found that by vitrifying (freezing a material such that it has a glass-like structure) biological substances, they were able to obtain extremely high-resolution microscopy images of these substances. This was the dawn of a technique which would have such a profound impact on imaging in structural biology that it would later earn Dubochet (and fellow scientists Joachim Frank and Richard Henderson) the 2017 Nobel prize in chemistry.

In recent years, the technique has become a precious tool in rational structure-based drug discovery. By accurately determining the shape of biological targets and how it changes in different biological contexts, scientists are much better able to predict what key structural features might be needed in a drug to fit these targets in different scenarios.

One hugely important area of drug discovery in which this technique is being utilised is the field of antibiotic resistance. Bacterial mutations can lead to alterations in the structure of protein targets of various drugs, leading to bacterial resistance to these existing drugs. Imaging and structurally determining these altered protein targets is giving drug discoverers the upper-hand in the fight to overcoming different types of drug resistance. Detailed imaging of the protein structures is like having a detailed map of the exact formation of an enemy army. While current strategies may not be the right fit to take them on, this secret information could be vital to help scientists adapt their approach and mount a new plan of attack.

Cryo-EM has become a vital imaging tool in recent years. However, the fast-moving field of imaging in structural biology is still evolving.

One specific challenge is how to determine the structure of a protein while it is in the intracellular environment. Proteins are highly dynamic and liable to change their shape depending on their environment. Therefore, while existing techniques can determine the structure of isolated proteins at high resolution, these structures may not be accurate representations of the shape of the protein when it is within or on a cell.

Being able to visualise proteins in situ at high resolution would be a promising next step to aid our knowledge of drug resistance mechanisms. Cryo-electron tomography (cryo-ET), a new twist to cryo-EM, could be the key to doing so. Cryo-ET uses the same TEM based imagery technique to cryo-EM. However, instead of single snapshots, cryo-ET collects series of images of rotating objects in situ to create more accurate 3D images.

The beauty of such brilliant techniques as cryo-EM and cryo-ET is that one single technique can provide a boost to scientists working to solve a plethora of global challenges.

At J A Kemp, we can’t wait for the exciting inventions that these techniques are going to keep bringing our way. Our specialists in Optics and Photonics have extensive expertise in protecting IP surrounding new and improved imaging techniques and can offer detailed advice in this area. J A Kemp’s Pharmaceutical and Biotechnology and Life Science teams specialise in protecting the new therapies and related inventions that such techniques make possible.