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

doi:10.1016/j.xpro.2021.100642

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

Department of Biosystems and Biotechnology

Research output: Contribution to journal › Article › peer-review

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).

Original language	English
Article number	100642
Journal	STAR Protocols
Volume	2
Issue number	3
DOIs	https://doi.org/10.1016/j.xpro.2021.100642
Publication status	Published - 2021 Sept 17

Bibliographical note

Publisher Copyright:
© 2021 The Authors

Keywords

Protein Biochemistry
Structural Biology
X-ray Crystallography

ASJC Scopus subject areas

General Neuroscience
General Biochemistry,Genetics and Molecular Biology
General Immunology and Microbiology

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10.1016/j.xpro.2021.100642

Cite this

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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).",

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AB - 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).

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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

Bibliographical note

Keywords

ASJC Scopus subject areas

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