X-ray Crystallography: Seeding Technique with Cytochrome P450 Reductase

• Published in: Bio-protocol Vol. 12; no. 21
• Main Authors: Zhang, Bixia, Lewis, Jacob A, Hazra, Rishi, Kang, ChulHee
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 05.11.2022; Bio-protocol LLC
• Subjects: Biology; Methods; X-ray crystallography; Protein crystal seeding; Structure disorder; Crystal dehydration; Resolution improvement; Cytochrome P450 reductase
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.4546

Cytochrome P450 reductase (CPR) is a multi-domain protein that acts as a redox partner of cytochrome P450s. The CPR contains a flavin adenine dinucleotide (FAD)-binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting domain. To achieve catalytic events, the FMN-binding domain needs to move relative to the FAD-binding domain, and this high flexibility complicates structural determination in high-resolution by X-ray crystallography. Here, we demonstrate a seeding technique of sorghum CPR crystals for resolution improvement, which can be applied to other poorly diffracting protein crystals. Protein expression is completed using an cell line with a high protein yield and purified using chromatography techniques. Crystals are screened using an automated 96-well plating robot. Poorly diffracting crystals are originally grown using a hanging drop method from successful trials observed in sitting drops. A macro seeding technique is applied by transferring crystal clusters to fresh conditions without nucleation to increase crystal size. Prior to diffraction, a dehydration technique is applied by serial transfer to higher precipitant concentrations. Thus, an increase in resolution by 7 Å is achieved by limiting the inopportune effects of the flexibility inherent to the domains of CPR, and secondary structures of SbCPR2c are observed. Graphical abstract.