Publication date: 17/01/2014
Last update: 17/01/2014
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If we can see something, it's because the wavelength of light rays can discern it. In other words, there is a minimum size for any any object below which we can not see details using visible light, simply because the wavelength of light rays is too great to define the details. It is like trying to outline a very detailed image on paper using a pen with a too thick tip . If someone who has not seen the original image watches the profile that we have done with this pen, he/she will have just an average idea of the image.
This is basically what happens with optical microscopy. No matter how many lenses we use, we can not go beyond a glimpse of the larger cellular structures (organelles) and not in all their details. But then ... how can we get by to visualize smaller structures, such as biological molecules?
The trick is to use a wavelength shorter than that of light, small enough to outline details. In the case of biological molecules X-rays are used, because whereas the wavelength of visible light is between 400 and 700 nm, the X-rays wavelength is only 0.1 nm, resolution enough to observe atomic level details in biological molecules.
But of course, such a minimal wavelength requires an approach different to optical observation, because its effect on objects that we want observe, in this case molecules, cannot be observed directly. Instead, what is done is to direct an X-ray beam to the molecule. The rays strike the structure and, depending on the arrangement and size of every atom present in the molecule, they are deflected. This deviation is captured on film and subsequently revealed. This is what is called a diffraction pattern, and it consists of a series of spots whose intensity and disposition depend on the placement and size of the different atoms.
However, for that pattern to be sharp, the molecule must be oriented and stationary with respect to the x-ray beam. If the molecule is in a liquid or a gas, it is not possible to use this technique, because in that state, molecules are constantly moving and changing orientation. Ideally, the molecule should be "frozen" in a position, without losing its structural features. To achieve this immobility, the molecule must be crystallized. In a crystal, many copies of the same molecule are arranged neatly in the space at solid state, allowing them to be "watched" by X-ray diffraction the arrangement of their atoms from many different angles.
So, once obtained the crystal of the molecule we want to stud , it is subjected to exposure to X-rays from different angles, thereby obtaining many diffraction patterns. It is the turn of the computers, which take these patterns and analyze them to build the three-dimensional model of the structure of our molecule. In general, an electron density map is obtained from the diffraction data, which is more or less a contour map that allows to superimpose the positions of the atoms composing the molecule and therefore, to determine its structure.
Actually, the hardest part of X-ray crystallography is to obtain a good crystal. The quality if the crystal must be extremely high, requiring an extreme purity of the molecule to be crystallized, along with determining the ideal conditions to achieve a crystal that reflects as closely as possible the natural structure of the molecule. These conditions can be very difficult to achieve, especially considering that there are molecules that show changing conformations depending on ligands , hydrophobicity, etc.
However, X-ray crystallography is a powerful ally that can get interesting data for biotechnology and biomedicine. Crystallography of biological molecules has allowed us to infer the function of many biological systems in terms of their interaction with other molecules in the environment, thereby serving to increase our understanding of biology. Without crystallography, structure and function of DNA would still be elusive - luckily, Rosalind Franklin was there exploring the potential of this technology in the 50s. With this technique, many enzymes have provided valuable information about their natural ligands, and it has been possible to design alternative ligands that interfere either positively or negatively in their function. Finally, knowledge of the structure of proteins of biotechnological interest can be used to design more efficient versions of them, increasing their industrial value and allowing the progress of many industrial processes.
Author: Vicente Díaz Martínez