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The benefits of a cryoEM

November 26th 2018

The Centre for Microscopy and Molecular Imaging (CMMI) has recently acquired a cryoEM. This low-temperature electron microscope can recreate the 3D structure of viruses, biological nanomachines, and proteins, at the atomic scale. It offers a number of benefits compared to traditional microscopy.

In order to be viewable, samples intended for a traditional electron microscopy must first undergo a series of treatments: chemical fixation in aldehydes, dehydration, coloration using heavy metals in order to increase contrast, embedding into resin for cutting, and so on. A cryoEM eliminates the need for all these operations!?‘Samples are directly immersed in a container of ethane cooled to -182 °C, itself placed in a vat of liquid nitrogen,’ explains professor David Pérez-Morga, head of CMMI's electron microscopy laboratory. ‘This “dual immersion” system cools the sample so quickly that the water molecules inside them do not crystallise, but simply become immobile. This means that the samples remain very close to their natural physiological state.’

Preparing proteins much faster
In order to visualise proteins at the atomic scale, traditional techniques requires processing large quantities of protein, making the samples as pure as possible, crystallising them, and subjecting them to ‘X ray diffraction’. This can be tedious and time-consuming, especially for proteins whose environment is not water, but fat. ‘It is very difficult to purify and crystallise those proteins in their physiological state,’ explains professor Pérez-Morga. ‘Working out the proper procedure can take up to twenty years… if it is ever done at all! Using the cryoEM, we can model hydrophobic proteins in a matter of months, from thousands of times fewer molecules, less pure samples, and with no crystallisation required.’

A camera that can ‘see’ proteins
The cryoEM’s major innovation, and the reason it won its inventors a Nobel Prize, is its ability to reconstruct atomic structures in 3D. ‘For instance, the image of a virus as captured by a traditional electron microscope is nothing more than the virus' shadow,’ explains professor Pérez-Morga. ‘As for the proteins that make up the virus, older cameras were not able to detect the signal caused by the proteins’ small atomic nuclei. The new sensors that the cryoEM uses, on the other hand, are sensitive enough to “see” these minuscule signals. Then, based on a series of 2D images of the virus, the software can reconstruct the atomic structure of all its proteins in 3D.’

Research prospects
One of the first proteins to be modelled using the CMMI's cryoEM will be apolipoprotein L1. Certain lab-designed versions of this human protein can destroy the parasites that cause sleeping sickness(2). ‘Being surrounded by a fat environment, apolipoprotein L1 cannot be crystallised. This is why we model it, in order to study it and understand how it interacts with several other proteins at a molecular scale. These interactions are also important in the development of certain kidney diseases in humans.’
Other research projects focus on proteins involved in resistance to antibiotics and other drugs. The cryoEM can also be used to model viruses and bacteria, as well as their protein components, and proteins involved in cancer processes.

Notes:
(1) In 2017, Jacques Dubochet, Joachim Frank, and Richard Henderson won the Nobel Prize in Chemistry for developing cryo-electron microscopy.
(2) Human African trypanosomiasis, also called sleeping sickness, is caused by a parasite carried by the tsetse fly.