Construct optimization for studying protein complexes: obtaining diffraction-quality crystals of the pseudosymmetric PSPC1–NONO heterodimer

The methodology of protein crystallography provides a number of potential bottlenecks. Here, an approach to successful structure solution of a difficult heterodimeric complex of two human proteins, paraspeckle component 1 (PSPC1) and non-POU domain-containing octamer-binding protein (NONO), that are involved in gene regulation and the structural integrity of nuclear bodies termed paraspeckles is described. With the aid of bioinformatic predictions and systematic screening of a panel of constructs, bottlenecks of protein solubility, crystallization, crystal quality and crystallographic pseudosymmetry were overcome in order to produce crystals that ultimately revealed the structure.