There are currently two main methods to determine the three-dimensional structure of a protein: Nuclear Magnetic Resonance (NMR) Spectroscopy and X-ray crystallography. While structure determination with the NMR method is limited to proteins with an upper molecular weight of approximately 25 kDa, the X-ray method is suitable to resolve the structure of larger proteins or macromolecular complexes.
Co-crystals of human TMP-kinase and 2'Br-ADP/ATP
The first step in the determination of an X-ray crystal structure, that is often also the most difficult one, is the growth of diffraction-quality protein crystals. Co-crystallization with protein specific ligands (e.g. substrates, cofactors, small-molecules) is already an integral part of most initial screening → Crystallography & Cryo-EM and optimization → Crystallography & Cryo-EM strategies.
For crystallization of enzyme-nucleotide complexes, non-hydrolyzable nucleotide analogs have become indispensable tools. This is due to their modified phosphate moiety that renders them resistant to hydrolysis and allows formation of stable nucleotide-protein complexes for structure determination[2,3].
Subsequent structure solution techniques (multiple isomorphous replacement (MIR) or multiple wavelength anomalous dispersion (MAD)) still involve the incorporation of heavy atoms into protein crystals. The search for suitable heavy-atom derivatives is the second major bottle neck in structure determination since binding of heavy atoms often results in disrupting the crystal lattice. Halogenated, mercurated and Selenium-containing nucleotides however, may provide an alternative method that allows rational incorporation of heavy atoms into a large number of nucleotide- or DNA-binding proteins.
 Hassell et al. (2007) Crystallization of protein-ligand complexes. Acta Crystallogr. D. Biol. Crystallogr. 63:72.
 Xia et al. (2011) Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase. Biochemistry 50:9114.
 Jiang et al. (2011) Use of chromophoric ligands to visually screen co-crystals of putative protein-nucleic acid complexes. Current Protocols in Nucleic Acid Chemistry 7.15.1-7.15.8:S46.