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

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 ther...

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Detalles Bibliográficos
Autores principales: Kim, Sulhee, Yoon, Ina, Kim, Sunghoon, Hwang, Kwang Yeon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260868/
https://www.ncbi.nlm.nih.gov/pubmed/34258600
http://dx.doi.org/10.1016/j.xpro.2021.100642
Descripción
Sumario: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).

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