Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization
Sulhee Kim1,2, Ina Yoon3, Sunghoon Kim3
1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea.
Abstract:
Leucyl-tRNA synthetase 1 (LARS1) synthesizes Leu-tRNALeu for protein synthesis and plays an important role in mTORC1 activation by sensing intracellular leucine concentrations. Here, we describe a protocol for the purification, reductive methylation, binding affinity measurement by microscale thermophoresis, T value measurement by Tycho, and post-crystallization soaking and cooling in cryoprotectants to improve crystallization of LARS1. Collectively, this allowed us to build the RagD binding domain, which was shown to be a dynamic region of LARS1 refractory to crystallization. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).
