Crystallization of Protein– DNA Complexes

Abstract

Determination of atomic resolution structures of protein– deoxyribonucleic acid (DNA) complexes using X‐ray crystallography requires preparation of high‐quality crystalline specimens. The preparation of such samples remains an empirical endeavour, more art than science. Nonetheless, decades of experience have yielded a large body of practical strategies and techniques towards this end. Such efforts with protein–DNA complexes enjoy special advantages, but also involve control of a larger set of variables. High purity, chemical and conformational, preparations of both macromolecules are important. Crystallogenesis of protein–DNA complexes is favoured by careful choice of the sequence and the terminal bases and/or base pairs of the DNA target. Modern crystallization screens, which encapsulate the accumulated wisdom of prior investigations, serve as an excellent starting point for experimentation. Key Concepts An atomic level analysis of a macromolecule by X‐ray crystallography requires preparation of high‐quality crystals of the entity of interest. Preparation of crystalline specimens requires highly concentrated solutions of the molecules of interest. Success is favoured when high chemical and conformational purity has been achieved. Extensive experience in preparation of protein crystals can be applied to growth of protein–DNA crystals. Additionally, the length, composition and identity of the termini of the DNA molecule play significant roles in crystallization. Initial crystallization trials are set up using a concentrated stock of protein–DNA complex of interest with pre‐made formulations that sparsely sample crystallization space. Precipitation and crystal formation under these conditions are used to direct efforts for further crystallization experiments. The experiments are repeated until crystals suitable for X‐ray diffraction are obtained.

Solvated protein–DNA docking using HADDOCK

“…The amount of structural information concerning protein-DNA complexes is increasing rapidly due to numerous efforts from the X-ray crystallography (Pakotiprapha and Jeruzalmi 2009) and NMR spectroscopy (Clore 2011;Varani et al 2004) fields and to computational advances in analysis (Dunn and Kingston 2007), modeling (Aloy and Russell 2006;Baker and Sali 2001;van Dijk and Bonvin 2010) and simulations (Giudice and Lavery 2002;Pérez et al 2012). In particular docking, a computational approach that models the unknown structure of a complex from its constituents, is a valuable tool to study complex formation in interaction networks (Melquiond et al 2011).…”