Structure elucidation by diffraction methods – contribution to structural biology


J. Dohnálek


Institute of Macromolecular Chemistry of the Academy of Science of the Czech Republic, Heyrovského nám. 2, 16206 Praha 6, Czech Republic


Keywords: protein crystallography, X-ray diffraction, structural biology


The first documented X-ray scattering on ordered samples of biological origin was observed by J. D. Bernal in Cambridge, U.K. in 1934. Further experiments by Dorothy Crowfoot-Hodgkin and M. F. Perutz in Oxford on crystals of insulin and haemoglobin in 1937 and in the following years lead to development of a method which revolutionized our views of biochemical processes and molecular structure of living systems. The number of structures determined by diffraction methods from 1960 to these days (the first one of myoglobin by Kendrew) amounts to tens of thousands, many of them remaining unpublished or subject to proprietary rights of private companies. The length of the process of solving and finalizing a structure dramatically decreased from several years to several weeks or months depending on the project difficulty.

While from 1950s till 1980s methods for structure solving such as molecular replacement and isomorphous replacement were developed, late 1980s and 1990s saw a fast development of computational technology and its application in all steps of macromolecular structure determination. In the recent ten years this field profited from huge development effort in crystallographic software as well as methodological advances in protein and nucleic acid crystallization and in data collection and analysis. Macromolecular crystallography today would be unthinkable without cryo-cooling of samples, synchrotron sources of high intensity X-ray radiation and high speed internet services and databases.

Crystallographically determined structures of biological molecules bring invaluable information ranging from details of interactions between ligands and enzymes, assemblies of protein-protein or nucleic acid – protein types to highly complex structures of viruses. Quality of acquired structural information does not depend on the size of the system once regularly organized.

The most recent advances (5 years) target the main bottlenecks of these methods and indicate future development. High throughput methods for protein production, purification and crystallization, robotized home source and synchrotron beam-lines and software equipment for automatic information-with-sample transfer and for automatic data collection were produced mainly within structural genomics projects for which the main starting impulse were the results of successful genomics projects. Systematic studies on radiation damage of biological samples induced by intensive radiation sources contributed substantially to better experiment planning and techniques.  Free electron laser and single particle imaging are becoming centres of interest mainly for diffraction studies of larger systems (organelles or cells) without the need of an ordered crystalline system.


Introduction of modern methods is supported by the Ministry of Education of the Czech Republic (1K05008).