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
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author Kim, Sulhee
Yoon, Ina
Kim, Sunghoon
Hwang, Kwang Yeon
author_facet Kim, Sulhee
Yoon, Ina
Kim, Sunghoon
Hwang, Kwang Yeon
author_sort Kim, Sulhee
collection PubMed
description 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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spelling pubmed-82608682021-07-12 Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants Kim, Sulhee Yoon, Ina Kim, Sunghoon Hwang, Kwang Yeon STAR Protoc Protocol 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). Elsevier 2021-07-02 /pmc/articles/PMC8260868/ /pubmed/34258600 http://dx.doi.org/10.1016/j.xpro.2021.100642 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Kim, Sulhee
Yoon, Ina
Kim, Sunghoon
Hwang, Kwang Yeon
Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
title Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
title_full Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
title_fullStr Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
title_full_unstemmed Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
title_short Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
title_sort protocol for improving diffraction quality of leucyl-trna synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
topic Protocol
url 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
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