Crystallographic structure of a chaperone protein (PDB 1AON10) -

© Thomas Splettstoesser / Wikimedia CC by-sa 3.0

  • Observation of proteins is essential because their dysfunction can be the cause of many diseases such as cystic fibrosis or Alzheimer's, according to a study published by our partner The Conversation.

  • One of the best tools to do this is the electron cryo-microscope (or cryo-electromicroscope), which is imaged at a temperature of -196 ° C.

  • The analysis of this phenomenon was carried out by Léni Jodaitis, doctoral student and assistant in chemical sciences (specialization in biochemistry) at the Université Libre de Bruxelles.

It is possible to obtain very detailed structures of proteins.

These molecules are made up of a few tens to hundreds of thousands of atoms and have complex chemical and 3D structures.

These macromolecules govern all the mechanisms of life at the cellular level.

Proteins are very important for biology and medicine.

True biological workers, proteins can perform very specific functions.

For example, antibodies are able to recognize foreign bodies.

Insulin, which is involved in certain metabolisms, regulates the absorption of glucose.

Or, “ion channels” allow molecules needed by our body such as salt and water to enter our cells.

Protein dysfunction can be the cause of many diseases such as cystic fibrosis or Alzheimer's.

Three-dimensional structure of a SARS-CoV-2 spike and its glycosylation sites.

Atomic resolution is achieved and valuable information can be extracted from this structure.

Knowing their chemical structures, at the atomic scale and in three dimensions, allows us to understand the functioning of the systems in which they intervene and to be able perhaps to correct, help or stop certain biological processes.

How can we hope to look at atoms under a microscope?

To observe elements invisible to the naked eye, humans have always been creative.

One of his inventions is the optical microscope.

Thanks to a lens system, it makes it possible to visualize objects of a few micrometers, or a thousandth of a millimeter, but this is not enough to see isolated atoms or constituents of a protein.

This limitation is related to the wavelength of visible light.

To observe atoms, several techniques exist.

Atomic force microscopy and tunneling microscopy use very fine tips that end

in fine

with a single atom.

These tips probe surfaces and detect atoms and / or molecules present.

However, the most popular technique in biochemistry has always been crystallography.

To obtain the structure from protein crystals, X-ray diffraction obtained in synchrotrons is used.

Three-dimensional structure of the 2019-nCoV spike protein obtained by cryo-electron microscopy.

© D. Wrapp, N.Wang, KS Corbett, JA Goldsmith, CL Hsieh, O. Abiona, BS Graham, JS McLellan - Science, 2020 - CC BY (via The Conversation)

Another solution is to use the same principle as the optical microscope, but with electrons rather than photons, because it is easier to manipulate their trajectories since they are charged particles.

This is called a transmission electron microscope.

This technique continues to improve over time and the scientists who developed it in 1933 were awarded the Nobel Prize in physics in 1986, along with the designers of the tunneling microscope.

The general principle remains the same: observe the interaction of electrons with the sample and deduce the surface or the shape of the object.

The electrons are emitted through a heated tungsten filament, then concentrated through magnetic fields rather than lenses.

The electrons are charged and their trajectory is deflected towards the sample by the applied field.

After interacting with the sample, the electrons encounter a detector, and the signal is processed to generate images of a previously invisible world.

This technique has a few flaws: it is particularly energy-intensive, requires a complete vacuum (there is no more air in the system at all) and the slightest variation in the magnetic field influences the image.

Its biggest limitations are thermal agitation and sample destruction.

When taking an image of such small items, even the slightest movement decreases the resolution (detail) that can be achieved.

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low temperature

It is also on this principle that the electron cryo-microscope (or cryo-electromicroscope) works: “cryo” for “cryogenic”, because all the imaging is carried out at a temperature of -196 ° C;

and “electronic” because the energy source for observing the sample is provided by electron radiation.

Working at such low temperatures reduces thermal agitation and protects molecules from destruction in order to achieve better resolution.

Video “

What is electron cryo-microscopy?

"(Source: CNRS)

A real revolution, in particular for applications in biology for which samples of organic matter are often too fragile to be imaged in a conventional transmission electron microscope, this tool earned its designers the Nobel Prize for chemistry in 2017.

The sample to be observed is first placed on a small grid a few millimeters in length, then it is frozen very quickly in order to avoid the formation of water crystals thanks to liquid ethane, which freezes in a few microseconds. the sample on the grid (“vitrification”).

The grid is then inserted into the microscope.

At this point, the images show black spots on a gray background.

The example used here is that of a membrane channel whose actual size is only about fifteen nanometers, or 15 millionths of a millimeter.

Photomicrograph of a sample containing a potassium ion channel protein.

To move from a collection of photomicrographic images (an example on the left) to a two-dimensional reconstruction and classification of objects (on the right), selection and superposition algorithms are needed, but above all a few hours of calculation © D. Matthies, C. Bae, G. ES Toombes, T. Fox, A. Bartesaghi, S. Subramaniam, KJ Swartz - eLife (via The Conversation)

Computer reconstruction to see the 3D structure of molecules

The power of the cryo-electron microscopy technique is due to the combination of imaging and the computer processing that follows.

From hundreds of thousands of images, it is possible to reconstruct a three-dimensional model of the structure of the object.

The more images, the better the resolution of the reconstruction.

Since the amount of data to be processed is large, with millions of 2D images for a single 3D structure, scientists resort to programs that use automated and guided processes.

The three-dimensional structure of the ion channel after the reconstruction is complete.

The scale on the right represents the resolution: around two angstroms, the atomic resolution is reached.

The higher the value of the resolution, given in angstroms (Å), the less detail we have © D. Matthies, C. Bae, G. ES Toombes, T. Fox, A. Bartesaghi, S. Subramaniam, KJ Swartz - eLife (via The Conversation)

The results are breathtaking.

For example, by observing the “spike” protein of SARS-CoV-2 with atomic resolution, we could quickly understand the process of glycosylation - the attachment of sugars to the



of the virus.

This process is important in the life cycle of the virus to be able to escape the human immune system.

A long history, recent technological developments, immense potential

Cryo-electron microscopy has been around for about fifty years and was initially called “blobology” because the results of the analyzes often only led to a shapeless mass that scientists call a “blob”.

Thanks to recent technological advances, electron cryo-microscopy is widely used to define the structures of the most complex proteins.

Recent improvements in detectors, electron sources, image processing methods and processors suggest avenues for improving the technique.

Our "Biology" dossier

It is now possible to discover systems hitherto unobservable under a microscope.

The details are such that the position of each atom that makes up the observed sample is made visible!

Only 50 years ago, atomic resolution in microscopy was just a physicist's dream and now it has become a reality.

Cryo-electron microscopy is certainly the technique with the most potential in meeting the next challenges of cell biology (antibiotic resistance, the discovery of new vaccines, cancer treatment, etc.).

Thanks to this technique, we will be able to better understand the functioning of our cells, but also bacteria and viruses.

This could potentially open up yet unexplored pharmacological opportunities and make it possible to cure diseases that currently affect humanity.


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This analysis was written by Léni Jodaitis, doctoral student and assistant in chemical sciences (specialization in biochemistry) at the Université Libre de Bruxelles.

The original article was published on The Conversation website.

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