Protein purification, crystallization, and structure determination of transcription factor YhaJ in complex with DNT metabolites

The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives. For complete details on the use and execution of this protocol, please refer to Kim et al.1 [Display omitted] •Instructions for purification and characterization of YhaJ effector-binding domain•Steps for crystallization of YhaJ with a DNT derivative•A step-by-step guide for structure determination of the YhaJ:DNT derivative complex Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives.