X-Ray Crystallographic Studies of G-Quadruplex Structures
The application of X-ray crystallographic methods toward a structural understanding of G-quadruplex (G4) motifs at atomic level resolution can provide researchers with exciting opportunities to explore new structural arrangements of putative G4 forming se
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ntroduction An understanding of molecular structures at atomic resolution has principally relied on the application of either X-ray crystallography or Nuclear Magnetic Resonance (NMR) methods, with crystallographic methods requiring the samples to be constrained in a solid state within an ordered crystalline lattice (Fig. 1). Unfortunately, the generation of suitable crystals containing ordered
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_8, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Gary N. Parkinson and Gavin W. Collie
Fig. 1 Path to structure determination by single-crystal X-ray diffraction methods. (a) (i) Crystal mounted in nylon loop in cryostream and exposed to X-rays. (ii) Resulting diffraction pattern with solvent ring around 3.5 A˚. (iii) Schematic drawing of final coordinates modeled into resulting electron density maps. (b) Pathway to crystallographic structure determination
G-quadruplexes (G4s) remains the main obstacle to the successful application of X-ray crystallography as a tool for the determination of G4 structures. However, when high-quality crystals are formed, and diffract to better than 5 A˚ resolution, then, with access to high flux tuneable synchrotron X-ray sources, fast computers, and automated software, the successful determination of the nucleotide structures should almost be guaranteed, certainly with target structures with molecular weights around 7–15 kDa (Figs. 1 and 2) [1, 2]. The requirement for the generation of crystals then imposes further experimental constraints on the oligonucleotides, such as chemical purity, homogeneity of sequence and topology, a limited selection of solvents, along with the imposition of constrained packing interactions for the DNA/RNA molecules. However, if you are able to generate crystals and the structure is “solved,” this provides a significant opportunity to the researcher to identify any ordered scattering component whose characterization only relies on correct interpretation of its electron density, and its context to other surrounding components. The methodology also allows for the visualization of ordered solvent directly, and critically for
X-Ray Crystallographic Studies of G4 Structures
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Fig. 2 Examples of G4 structures determined by single-crystal X-ray diffraction methods (PDB-ID codes given). Purple spheres represent Na+ ions (a, b) or K+ ions (c–f) and green spheres represent Ca2+ ions (a, b) or Mg2+ ions (d)
quadruplex structures, the metal ions in the central channels, the waters or any other associated elements essential to G4 formation. Extending further, the determination and interpretation of the associations of any additional components added to the crystallization buffer, such as small molecule ligands, can be visualized and their interactions to the quadruplex target identified without bias derived from our predetermined expectations. For example, even subtl
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