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Half a century of proteins through glass

The 50th anniversary of the radiological analysis of the molecular structure of proteins in three dimensions is being celebrated. Max Perutz and John Kendrew started with myoglobin and hemoglobin. Today there is now a database with the identification of the structure of about 50,000 proteins. And Michael Rossmann, a pioneer in molecular crystallography, ensures that many more remain to be identified. Recently the seminar '50 years of protein structures' was held, organized by the Institute of Institute for Research in Biomedicine (IRB Barcelona), commemorating the first half of the century of the discipline.

Jordi Montaner | 18 February 2010


Photo: Thomas Splettstoesser
X-ray diffraction to study the structure of protein crystals requires the involvement of unique crystals, of perfect quality and with no more than one conformation. Many macromolecules have failed because they present very hydrophobic portions (they do not absorb water easily) that cannot be crystallized. It turns out that each diffraction pattern consists of thousands of signals that indicate the position and intensity at each point; the phases of the waves at each point provide in this way, an electron density map.


Each application of the rays on the crystal under investigation provides data concerning the position, intensity and phase, configuring what is called 'structure factor'. It is not easy. Celerino Abad-Zapatero, a researcher from Burgos (Spain) who works with Michael Rossmann at Purdue University (Indiana, United States) and was present at the seminar, details that the Protein Data Bank (PDB) contains structure factors of only 25% of the crystallographic entities tested. By publishing the structure factors, hence, other researchers can generate and use density maps that are much more useful.

Each application of the rays on the glass provides data on the position, intensity and the phase "The X-rays are diffracted by electrons from molecules in the crystal. Hence, the results of a crystallization performed successfully can be configured as an electron cloud (electron density maps)”. This image, explains Abad-Zapatero, allows researchers to construct a model of the protein adjusted to the map obtained before. "The resulting pattern of amino acids or nucleotide sequence is then adjusted to the electronic density map, refining the structure with a set of Cartesian coordinates (x, y, z) for each non-hydrogen atom.


From the technique the molecular 'disorder' is assessed, i.e. the different positions occupied by the atom in the molecule. We know that when there is disorder in the portions of a chain, poor or not very homogenous electron densities are produced, making it difficult to assign positions to the atoms.

A booming science

The development of structural crystallography is discarded by so many vicissitudes which makes it closely linked to advances in other areas of science and technology: chemistry, physics, mathematics, computer science and robotics.

As much as the German Wilhelm Roentgen made public his discovery of X rays in 1895, the use of radiation to determine the crystal structure did not spread until 1912, when Max von Laue found that a crystal exposed to X-ray beam
rises to a series of mysterious shadows of interpretation. In 1934, John Desmond Bernal and Dorothy Crowfoot Hodgkin discovered that crystals of pepsin produced excellent diffraction patterns, but were unable to interpret the data.

It was not until two decades after, Max Perutz in Cambridge, took advantage of this knowledge to investigate the structure of hemoglobin, an organo-metallic complex, where he replaced an iron atom by another one of mercury. Perutz continued to work with this mutant hemoglobin whilst John Kendrew, his disciple, determined with the same method the tridimensional structure of myoglobin. Both researchers were honored in 1962 with the Nobel Prize for chemistry. The same year, Watson, Crick and Wilkins received the Nobel Prize for physiology and medicine for uncovering the structure of various proteins and in particular that of the DNA. All the winners belonged to the so called Cavendish study group, of which Rossmann was part of and who, half a century ago, gave biomedicine some of his most important findings.

From structure to function

Over the years, new technologies have become available to crystallographers with more and better resources to analyze the results of X-ray diffraction in protein crystals.

After having solved the riddle of the structure, those of the function were left to be clarified. Currently, researchers at multiple centers, including the IRB Barcelona, are trying to assign a function to each of the new genes identified in the human genome in relation to a particular protein, elucidating its three-dimensional structures. "We think the function of proteins is closely related to its three-dimensional structure”, justifies Abad-Zapatero. The researcher adds that another application of the knowledge of three-dimensional structure of proteins is the design of new drugs or studies commissioned to draw evolutionary sequences and predict future structures.

"Today, in a protein crystallography laboratory equipped with a synchrotron of third generation and a robot controlled by computer, it is possible to mount, focus and expose protein crystals against a radiation source and obtain reliable results in less than
24 hours”. Abad-Zapatero explained that 50 years ago this meant, several weeks of hard work and which was not always rewarded.

A RAY OF LIGHT In physics, diffraction is a phenomenon that involves the scattering of waves when they encounter an obstacle. It happens with all kinds of waves: acoustic, the surface of a fluid, electromagnetic like light or of radio frequency. In the case of protein crystals, diffraction occurs when light encounters a certain structure, similar to how a light beam is deflected when colliding with the slit of a plaque.

Very recently, the use of a synchrotron allows obtaining structural information much more quickly and accurately. The identification of the relative position of atoms to light passing through glass provides information on the mechanisms that a protein uses to carry out some functions.

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