Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants

Sulhee Kim, Ina Yoon, Sunghoon Kim, Kwang Yeon Hwang

Research output: Contribution to journalArticlepeer-review

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, Ti 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).

Original languageEnglish
Article number100642
JournalSTAR Protocols
Volume2
Issue number3
DOIs
Publication statusPublished - 2021 Sep 17

Keywords

  • Protein Biochemistry
  • Structural Biology
  • X-ray Crystallography

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Neuroscience(all)
  • Immunology and Microbiology(all)

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