Expression, purification and crystallization of phyB and characterization of putative PIF1 from Sorghum bicolor

The main aim of this study was to investigate the structural differences between the photosensory modules of plant phytochromes and cyanobacterial phytochromes. To this end, three constructs of Sb.phyB, namely NPGP, PGP and PG, were successfully cloned, produced, purified and crystallized. Unlike prokaryotic phytochromes such as Cph1, obtaining diffraction-quality crystals for plant phytochromes is extremely challenging, indeed, no structure for a complete phyB photosensory module has been published to date. In this study, however, it proved possible to generate both NPGP and PGP crystals diffracting to 6 Å. PG crystals diffracting to atomic resolution were also generated and a complete dataset was collected, albeit with some anomalous pattern. SER mutation and additive screening efforts were also completed to mitigate difficult crystal morphology and improve the diffraction quality of NPGP and PG crystals. Additional work is still required to solve the structure of these fragments.