Protein crystallography is one of the key techniques of structural biology and is focused on determination of three-dimensional structure of proteins or nucleic acids by the means of single crystal x-ray diffraction. As proteins are both structural and functional units of living organisms the knowledge of their structure helps our understanding of their mechanisms and functionality and also enables targeted modifications with effects on their activity. New information acquired by this technique leads to explanation of the basic principles of functions of living organisms and at the same time to new approaches in fight against diseases such as cancer or AIDS and to many industrial biotechnology-based applications.
Samples of bio-macromolecules
The process of structure determination of a protein begins by production of a protein sample of sufficient amount (milligrams) and quality. The procedure is rather complicated and time-consuming. Our protein samples for structure analysis are usually produced by collaborating research groups.
Protein crystals are macromolecular crystals with a large solvent content
The next step if protein crystallisation. Compared to inorganic or organic compounds protein crystallisation is much more complicated and is performed in aqueous solutions; a resulting crystal exists in an equilibrium with the “mother” solution and approximately one half of its volume are channels filled by unordered solvent. The process depends on many parameters, e.g. protein and precipitant concentration, temperature, pH, purity and homogeneity of protein sample, crystallisation method and experiment set-up, etc. There is a large number of parameters influencing protein crystallisation and it is practically impossibility to forecast the results or simulate the process. This often makes obtaining of a crystal suitable for diffraction experiments time-consuming and experimentally demanding. Search for suitable crystallisation conditions for one project can take several weeks to several years.
\Mere visual evaluation of quality of protein single crystals is insufficient and their suitability for structural studies is always tested properly only by an x-ray diffraction experiment. Frequently the quality of diffraction is not sufficient for structural analysis and varies with crystals. Therefore a large number of crystals of different morphology must be quite often screened with concurrent optimization of crystal cryo-protection.
Diffraction experiment
Size of unit cell of protein crystals varies between tens and hundreds of Ångström in one direction and together with a relatively higher proportion of unordered atoms results in much weaker intensity of x-ray diffraction patterns when compared for instance with inorganic single crystals. Therefore more intensive sources of x-ray radiation are preferred such as a rotating anode or synchrotron radiation sources. To limit the extent of radiation damage to protein single crystals the diffraction experiments are usually carried out at low temperatures (80-120 K). This is achieved by a controlled stream of vapours of liquid nitrogen in majority of the cases. The so called oscillation method is most commonly used for diffraction data collection.
Protein diffraction experiment with Oxford Diffraction Ultra Enhanced Gemini system
Phase problem
To determine the three-dimensional structure of a given molecule once the diffraction intensities are measured it is necessary to determine or at least estimate initial values of phases of structure factors. In the case of macromolecular crystals two basic approaches are used: either similarity of the studied structure with a known one is exploited (molecular replacement) or a set of initial phases is determined experimentally, for example by means of anomalous dispersion of heavier atoms present in the molecule (e.g. MAD – Multiple wavelength Anomalous Dispersion).
In some cases structure solution (determination of initial phases) can be a lengthy procedure requiring persistent experimental work (e.g. years of search for suitable crystal form and at the same time heavy atom derivatives of such crystals). Direct incorporation of heavy atoms in native or engineered protein form makes experimental phasing easier. Direct methods of structure solution (computational solution of the phase problem) have been successfully applied with some smaller proteins providing diffraction data to atomic diffraction limits (resolution). There are two reasons why such approach cannot be applied routinely: limited resolution of x-diffraction of most protein crystals and mathematical dimensionality of the computational task (thousands to tens of thousands of non-hydrogen atoms forming one asymmetric unit of a crystal).
Protein structure refinement
Refinement of protein structures is carried out computationally and manually with use of specialized software designed for this purpose. Depending on structure complexity and data quality this step lasts weeks to months. A finalised crystal structure of a biological macromolecule is validated as for its agreement with experimental data and with expected stereochemical parameters and then deposited together with its experimental data in the international databank of protein structures PDB (Protein Data Bank, http://www.rcsb.org/). Both structural and experimental data are publicly accessible.
Example projects - Small laccase from bacterium Streptomyces coelicolor
One oscillation diffraction image recorded at a synchrotron radiation source with the above crystal of small laccase.
Symbolic representation of the trimer of small laccase from Streptomyces coelicolor. The enzyme forms compact trimers – each colour represents one covalent protein chain, orange spheres are copper ions. The enzyme oxidizes phenolic substrates and transfers electrons to the trinuclear cluster site where molecular oxygen is reduced to water. The function of the enzyme fully depends on the presence of Cu ions which also – in this case – contribute significantly to the stability of the trimeric arrangement. Individual atoms of the protein chains are not displayed for clarity – the trimer accounts for coordinates of about 12,000 atoms.
Packing of SLAC trimers in “crystal“. Individual molecules are distinguished by colours, the remaining space is filled by water and polymeric precipitant.
A close-up view of the substrate binding site of the SLAC trimer: phenolic substrates are supposed to bind approximately in the position of the depicted water molecules (orange spheres). Electron originated from substrate oxidation is transferred via copper ion Cu1 further down towards the trinuclear cluster of copper ions. The trigonal shape of the substrate binding site was observed for such enzymes for the first time.
Skálová T., Dohnálek J., Ostergaard L. H., Ostergaard P. R., Kolenko P., Dušková J., Štěpánková A., Hašek J.:
The structure of the small laccase from streptomyces coelicolor reveals a link between laccases and nitrite reductases.J. Mol. Biol. 385, 1165-1178, (2009).
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