Background

Cytochrome P450 reductase (CPR, EC 1.6.2.4) is located at the cytosol side of the endoplasmic reticulum membrane in eukaryotic cells, acting as the electron donor to cytochrome P450s and other heme proteins (Phillips and Langdon, 1962). It has been known that there are one to three CPR genes in most vascular plants (Jensen and Møller, 2010). The CPR contains a flavin adenine dinucleotide (FAD)/NADPH-binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting (or linker) domain with a flexible hinge region that links these two domains (Munro et al., 2001; Wang et al., 1997). We have reported on the structural flexibility of sorghum CPR isoforms (Zhang et al., 2022b) and the rapid interdomain opening and closing motion that resulted in fast interdomain electron transfer in SbCPR (Zhang et al., 2022a). Especially in the SbCPR2c isoform, which was crystallized in full-length, the FMN-binding domain was not resolved due to the aforementioned flexibility. Such flexibility causes uncertainty of the residue atomic coordinates and dihedral angles of the protein crystal, which leads to local disordered structure and overall low-resolution diffraction.

Crystallization of diffraction quality crystals is the largest bottleneck in protein structure determination (Holcomb et al., 2017). These difficulties have been overcome through the use of various techniques. Seeding uses previously nucleated crystals to initiate the growth of larger crystals in a fresh drop where protein concentration has not yet been depleted by the many nucleation sites of the small crystals (Rhodes, 2006). Micro streaking operates by running a fiber over an existing crystal—often a whisker from an animal—through a fresh drop to increase growth of the nucleated particles in a supersaturated drop (Stura and Wilson, 1991). In contrast, a macro seeding technique utilizes larger crystals, and thus requires more precision as a chosen crystal is washed and transferred from its original drop to a new mother liquor. This method tends to be more meticulous and can fracture the original crystal, causing smaller fragments to be placed into the new drop, which induces micro seeding (Zhu et al., 2005). Seeding techniques have allowed for growth of larger crystals and increased resolution of diffraction; cellobiohydrolase II (CBHII) crystallization was first achieved by using seeding to avoid formation of microcrystals and diffraction greater than 2.0 Å (Bergfors et al., 1989). High mosaicity and cracking/sliding of the lattice caused by impurities have been overcome by using seeding to reduce crystal deficits and improve the resolution of crystals (Caylor et al., 1999). Another method for improving crystal lattice packing is the dehydration of the crystalline to reduce water contents and to consequently confer tighter packing (Timasheff, 1995). Dehydration can be accomplished via exposure to the atmosphere or via serial dilution into higher cryoprotectant-containing solutions (Heras and Martin, 2005). Crystal dehydration has been one of the most successful post crystallization treatments in increasing resolution. Notably, the resolution of disulfide bond isomerase was improved from 7 to 2.6 Å and from 12 to 2.6 Å in E. coli YbgL (Abergel, 2004; Haebel et al., 2001).

We incorporated a macromolecule seeding technique—additive solution optimization—together with dehydration to improve the resolution of the SbCPR2c crystal structure from approximately 12 to 4.5 Å. This enabled the determination of the secondary structure for SbCPR2c. It is worth noting, however, that crystal growth is a very tedious process with repeatability differing in trials due to miniscule changes of parameters and the complexity of the system. Despite the aforementioned difficulties, our protocol of seeding and dehydration could be widely applied to resolve the resolution problems to a variety of crystals hindered in resolution by their dynamic disorder.