Richard Henderson: Nobel Prize in Chemistry
The winner of the Nobel prize in chemistry 2017 was told about e-cryomicroscopy
Electronic cryomicroscopy allows for more efficient use of the possibility of such a laboratory tool, as the electron microscope. About it it would be impossible to even think before the discovery of electrons, which was made in the early twentieth century Joseph Thompson – recently we celebrated the centenary of the discovery of the electron. And once it became known that due to wave-particle duality of the electrons could be focusing just as light waves, the Ernst Ruski had the idea of creating an electron microscope. Its great advantage lies in the fact that the wavelength of the illuminating field of electrons in a hundred thousand times smaller than the same feature of the light beam. If the wavelength of light is 1 micrometer, the wavelength of electrons is measured by the PM.
In principle, this allows you to get a much higher resolution, but the technical side of this issue has been fraught with many difficulties. Only in the 1950-1960-ies managed to get the resolution of the electron microscope level atoms. The use of this technology biologists always remained behind, because the researchers used it primarily for the study of metals. The reason for this is that when you hover the electron beam on a biological object, consisting of organic molecules, the structure of this object is destroyed radiation exposure. In all of the first attempts to use electron microscopy to study biological objects the samples were destroyed. So originally all methods of use of electron microscopy techniques for solving biological problems was to introduce into the biological structure of heavy metals, in imitation of metallurgists and other researchers in the field of materials science.
This state of Affairs continued until the study did not take American scientist Bob Glaser, still working in Berkeley. He pointed to damage from radiation. This means that the electrons passing through the sample, break the connection and change its structure, turning it from protein, lipids and so on in the burned product. In the structure damaged by the hydrogen atoms, they are in the form of gas, and the researcher only remnants remain from the original structure. Glaser drew attention to the fact that it is impossible to determine the structure of individual molecules due to radiation exposure. And he suggested the use of crystals to investigate how these structures interact with the electrons.
If you have a structure consisting of ten thousand molecules, you can get information about it, even if you destroy several molecules. Glazer and his students at Berkeley was the first one tested the hypothesis that overcome or reduce the damage from radiation by cooling of the sample. And they were experimental samples cooled to -100 °C. This lower limit, which could be reached as electron microscopes were very poor vacuum chamber and they could be contaminated.
In 1980, appeared more sophisticated vacuum chamber, cold subject tables (to cool the sample to low temperatures), which allowed to reach the temperature of liquid nitrogen. It's made in the European laboratory of molecular biology Jacques Dubose in Heidelberg. There for three to five years, studied the properties of water, which is the normal environment for all biological structures. It was shown that by freezing the water forms ice crystals, sometimes hexagonal, cubic sometimes, but if you freeze water very quickly, it forms amorphous ice. And Jacques Dubose have developed a method to make a thin film of ice, by placing the sample in specific conditions. Initially, for these purposes, tried to use liquid nitrogen, but due to the fact that the temperature was close to the boiling point and turned the gas cooling was too slow, and formed crystals.
Now using a different approach, which is also invented by Jacques Dubose. It lies in the cooling liquid ethane or propane at a temperature of liquid nitrogen, but they remain liquid. When the liquid ethane is at its freezing point, and you layer on a thin film of water, there is a gap in 100 °C between the freezing point and the boiling point of liquid ethane. It turns water into a very thin film of amorphous ice.
I think that this is the beginning of an era of cryomicroscopy associated with the works of Dubose. At that time, we worked on two-dimensional crystals of protein called bacteriorhodopsin. We worked at room temperature, and cooling the samples to the temperature of liquid nitrogen, we could get 4-5 times more information to the moment will reach the level of the damage of radiation. That is, the cooling of the samples does not protect against the effects of radiation, but slows down the onset of the effects is 4-5 times. And so we used cryomicroscopy to determine the structure of 2D crystals.
The real power of e-cryomicroscopy is that it is possible to freeze anything: pieces of cloth, a solution of molecules, suspensions of viruses, it is possible to freeze the ribosome, virtually any object. Now in e-cryomicroscopy used four or five different approaches to different types of samples, each of which has its own interesting and sheds light on the different areas of biological knowledge.
The easiest way to freeze a suspension of single particles. For example, if you take ribosomes – site of protein synthesis – from different types of cells, it is possible to freeze a thin film with ribosomes and see all of the ribosome individually sealed in different positions. You can choose the ribosome, if they are present in sufficient quantity 10 or 20 thousand, averaging all views and make a 3D model of the structure.
The same principle applies in imaging, for example, when the patient discovered a brain tumor and he goes to the examination. Carried out x-ray irradiation from six different angles, and recovers the 3D picture of the brain, eyes and swelling. In structural biology, we do the same thing using electronic cryomicroscopy. You can explore a single particle, without any symmetry, but we need to get images from all possible angles.
Other type of samples in structural biology is to some extent a symmetric samples. For example, many viruses are spherical particles with means icosahedral symmetry. This means that there are double, triple, and five-part axis of symmetry that pass through the particle, and each means icosahedral particle contains 60 copies, 60 different angles. You get the picture means icosahedral particles from one angle, but actually in this case you will see particles that form the virus, with 60 different angles. Thus, to build a 3D model of the structure of this virus, it will take 60 times fewer images than for asymmetric particles of the same size.
You can also allow a lot of structures in biology, with a spiral organization, such as the alpha helix of actin filaments, which form muscle tissue. Muscle is a thin and thick filaments, they create tension in opposite directions, and the muscle shortens. It can be good to learn with the help of electronic cryomicroscopy: see the spiral filament to get the images and averaging them. Bringing an average structure, you need to consider different geometries: for a single particle it is necessary to accurately determine the orientation of helices predicted there is a geometric arrangement of one subunit relative to the other.
Next difficulty level of the organization is a two – dimensional crystals, in the study which used a single layer, but there's one direction for one axis and the other is for a - and b-axes, and this is called electron crystallography. Initially, this trend was more simple, because one crystal can consist of up to 10 thousand molecules in the same orientation.
In the end spirals out of single particles and two-dimensional crystals to learn common structures. In this case, the researchers used electronic cryotomography. This means that one sample, in which there is recurring and multiple structures, it is necessary to photograph from different angles in increments of plus or minus 70 degrees and to get the picture, just like in the case of brain tumor. And in this way you can study a range of different samples.
Using these techniques, the researchers were able to resolve the structure of hundreds and thousands of different molecules that could not be decrypted by other methods, such as x-ray crystallography or NMR spectroscopy. Over the past three years, there have been significant technical improvements, computer programs have become more powerful, and a new generation of direct electronic detectors has practically appeared. Instead of using films, we can now use the new solid state in silicon-based devices. This improves the signal–to-noise ratio when receiving images. So now we are living in an era of revolution in resolution, as Werner Kuhlbrandt called it in 2014.
Until 2010, it was possible to get large structures with high resolution, small structures, but they were like drops or blots. Biologists engaged in electronic cryomicroscopy were somewhat humiliatingly called blobologists (from the English blobs – "blots"), but now the time has come for a revolution in resolution, and all structural biologists are striving to use electronic cryomicroscopy. Today it has become a very powerful method, and people who have no experience in electronic cryomicroscopy enter this scientific branch. More and more research departments, universities, and research institutes are investing in electronic cryomicroscopy. Now there are not enough specialists to serve all possible areas that we would like to develop. Therefore, we can say that electron cryomicroscopy has become a very powerful method, perhaps the most important in structural biology.
About the author:
Richard Henderson – Professor of Molecular Biology, University of Cambridge.
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