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

ABSTRACT: Leucyl-tRNA synthetase 1 (LARS1) synthesizes Leu-tRNA^Leu 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 _i 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).

SUBMITTER: Kim S

PROVIDER: S-EPMC8260868 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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

Kim Sulhee S Yoon Ina I Kim Sunghoon S Hwang Kwang Yeon KY

STAR protocols 20210702 3

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 i value measurement by Tycho, and post-crystallization soaking and cooling in cryoprotectants to improve crystallization of LARS1. Collectively, this allowed u ...[more]

PMID: 34258600

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