THE HISTORY OF THE SCIENTISTS RECEIVED THE NOBEL PRIZE IN CHEMISTRY

After three years earlier the Nobel Prize in chemistry was awarded to three scientists for their contribution in the development of a high-resolution fluorescence microscope, this year's Nobel Prize chemistry was awarded to three scientists for their services in the development of molecular imaging techniques.

Jacques Dubochet, Joachim Frank and Richard Henderson, each from Switzerland, the United States (German-born), and Britain were awarded the chemistry Nobel Prize in 2017 for their contribution in the development of cryo-electron microscopy techniques or commonly shortened cryo-EM for biomolecular imaging with high resolution.

Jacques Dubochet, Joachim Frank and Richard Henderson for the development of cryo-electron microscopy, which both simplifies and improves the imaging of biomolecules. This method has moved biochemistry into a new era. A picture is a key to understanding. Scientific breakthroughs often build upon the successful visualization of objects invisible to the human eye. However, biochemical maps have long been filled with blank spaces because the available technology has had difficulty generating images of much of life's molecular machinery. Cryo-electron microscopy changes all of this. Researchers can now freeze biomolecules mid-movement and visualize processes they have never previously seen, which is decisive for both the basic understanding of life's chemistry and for the development of pharmaceuticals. Perhaps many new friends heard this one microscopy technique. Basically, this technique uses an electron microscope as in general, but the way the sample preparation and image processing approach becomes a completely different three-dimensional image. [1]

Only by knowing the atom-by-atom arrangement of a biomolecule can researchers grasp how it works — how, for instance, the ribosome reads strands of messenger RNA to manufacture proteins, or how molecular pores flip open and shut. For decades, one technique enjoyed a near monopoly in elucidating protein structures to this level of detail: X-ray crystallography, in which scientists persuade proteins to form into crystals, then blast X-rays at them and decipher the protein’s structure from patterns that the X-rays make when they bounce off. [2]

Before cryo-EM was developed, more commonly used for molecular imaging (generally a protein) was x-ray crystallography combined with data from NMR spectroscopy (Nuclear Magnetic Resonance). These techniques can indeed provide images of several molecules with high resolution but many fundamental limitations that make this technique unusable for all biomolecules.

Biologists are now pushing the technique further to deduce ever more detailed structures of small and shape-shifting molecules — a challenge even for cryo-EM. “Whether you call it a revolution or a quantum leap, the fact is that the gates have opened,” says Eva Nogales, a structural biologist at the University of California, Berkeley. [3]

X-ray crystallography requires that samples be converted to crystals (repetitive regular structures) first whereas not all molecules can form crystals. NMR spectroscopy can indeed provide supplemental information about the molecular structure and how a molecule interacts with other molecules, but only proteins of relatively small size can be sampled.

The story of this cryo-EM technique originated from Richard Henderson who, while earning his doctorate in 1960, used x-ray crystallographic techniques to obtain a protein image. Departure from that he realized the many shortcomings that must be covered to obtain images with high resolution while maintaining the original state of biomolecules.

In 1970, after many years of encountering many obstacles with x-ray crystallography, he eventually turned to an electron beam electron beam (an accelerated electron that has light-like properties).

Theoretically, the electron beam has a wavelength shorter than ordinary light so it has the potential to provide a high resolution to the atomic level. But in fact, electrons being bombarded into biomolecule samples can damage the sample so that all that can be detected is just the molecules die.

On the other hand, decreasing the intensity of electrons will produce less good images and tend to blur. In addition, because the electron microscope works in a vacuum state, the biomolecule sample will be damaged because the water around it evaporates so that the biomolecule is not in its original shape

But all began to see the bright point when Henderson using bacteriorhodopsin as a sample. Bacteriorhodopsin is a protein found in membrane organisms that can photosynthesize. Henderson and his team tried to take the image of bacteriorhodopsin without removing it from its original membrane. To prevent the sample from drying out, they coat the surface of the sample with a glucose solution.

After many trials, in 1975 a three-dimensional image of bacteriorhodopsin from various angles was obtained. In those days, this image was the best for a protein with a resolution of about 7 Å. Not satisfied at this point, Henderson continues to try to make the resolution of his image equivalent to the molecular image of x-ray crystallography of about 3 Å.

In Henderson's experiments, he and his team coated a sample of bacteriorhodopsin with a glucose solution. But here is a problem: water-soluble biomolecules can not be sampled using this method.

He couldn't coax proteins embedded in a cell membrane to crystallize. So he placed a bacterial cell membrane containing a protein called bacteriorhodopsin into an electron microscope and covered it in a glucose solution to keep it moist. He then lowered the amount of energy in the beam, creating a low-contrast picture. Because the molecules were embedded in the membrane in an orderly fashion, Henderson got a diffraction pattern that he could turn into a higher resolution image. By 1975, he had created a 3D picture of the protein. It was, at the time, the finest ever portrait made of a protein with an electron microscope. It was a bit of a special case because of the ordered arrangement of the proteins in the cell membrane, but in principle, this approach could be used for any molecule found in cells. [4]

The researchers tried to use other means by freezing the sample because ice evaporated more slowly than water. But what happens is the ice crystals dissolve the electron beam so that the image obtained is very far from expectations.

This is where Jacques Dubochet plays an important role. It has the idea of converting water into non-crystalline solids such as glass (vitrification) so that the electron beam can decompose uniformly and produce a uniform background.

In the early stages, Dubochet and his team sought to convert the water into the non-crystalline solid by freezing it in liquid nitrogen rapidly so that there was no time for water to form a crystal structure. In 1982, he and his team finally managed to get the non-crystalline water by firing it into cooled ethane in liquid nitrogen. This is what makes the cryogenic name (associated with low temperature) is embedded in this technique.

After that, they developed the technique so that it could be used for electron microscope samples by placing water-soluble samples on a kind of sieve so that the sample solution becomes a thin layer between the nets which then the filter is fired into ethane already cooled with liquid nitrogen. Now, the tool for vitrification of this sample is packed into a machine called vitrobot.

Then what is the contribution of Frank in the development of this cryo-EM technique? After Henderson and his team succeeded in obtaining a three-dimensional image of bacteriorhodopsin which in its original state has a uniform direction as in the picture, the researchers think how this method can be applied to all proteins and biomolecules primarily scattered in erratic directions in the sample.

The vitrification technique developed by Dubochet allows the sample to be frozen rapidly so that the biomolecules scattered within the sample become frozen in various directions and even in the middle of the process.

This allows a two-dimensional image of a biomolecule taken from multiple points of view. Joachim Frank and his team developed an algorithm that made it possible to combine two-dimensional images from different angles to produce a three-dimensional image.

Although all the fragments of this cryo-EM technique have been collected, in the early days the biomolecule image generated from cryo-EM is far from the resolution that x-ray crystallography can provide. In fact, in 1991 when Frank tried to take a three-dimensional image of the ribosome, he only got a rough contour that resolved around 40 Å, very far from the x-ray crystallographic resolution.

But along with the development of electron microscopy, the cryo-EM technique is also growing and more protein and other biomolecules that can be enshrined in the structure of the atomic level. Some of these are the capsid of the Zika virus, ribosomes, and thousands of proteins in the body of the organism.

Beginning with Henderson and bacteriorhodopsin, then refined by Dubochet with vitrification method of water and Frank with a two-dimensional image combining algorithm, the three of them succeeded in developing the cryo-EM technique so it can be used to get the image of the biomolecule with atomic resolution.

This brought about a major change to the world of biological science because eventually many proteins and other biomolecules that had not previously imagined how their shape and details could become visible in the atomic resolution are increasing with time. Whereas at first, the imaging of biomolecules with electron microscopes much regarded by other scientists.

Thus, the hard work and tenacity of these three scientists revolutionized the world of biomolecule imaging so as to bring many benefits to many other scientists. Especially Henderson, who always thinks positively that electron microscopy will continue to grow until it can provide an atomic-level image of the biomolecule.

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Refference and EndNote:

1. Jacques Dubochet, Joachim Frank and Richard Henderson for the development of cryo-electron microscopy

2. The atom-by-atom arrangement of a biomolecule can researchers grasp how it works

3. Biologists are now pushing the technique further

4. Chemistry

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