Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants
Summary: 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 microsca...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S266616672100349X |
| _version_ | 1819138132298170368 |
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| author | Sulhee Kim Ina Yoon Sunghoon Kim Kwang Yeon Hwang |
| author_facet | Sulhee Kim Ina Yoon Sunghoon Kim Kwang Yeon Hwang |
| author_sort | Sulhee Kim |
| collection | DOAJ |
| description | Summary: 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). |
| first_indexed | 2024-12-22T11:01:54Z |
| format | Article |
| id | doaj.art-f1b698979a9e4619a69c5053f7a98670 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-22T11:01:54Z |
| publishDate | 2021-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-f1b698979a9e4619a69c5053f7a986702022-12-21T18:28:28ZengElsevierSTAR Protocols2666-16672021-09-0123100642Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectantsSulhee Kim0Ina Yoon1Sunghoon Kim2Kwang Yeon Hwang3Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea; Institute of Life Science and Natural Resources, Korea University, Seoul 02841, Republic of Korea; Corresponding authorMedicinal Bioconvergence Research Center, Institute for Artificial Intelligence and Biomedical Research, College of Pharmacy & College of Medicine, Gangnam Severance Hospital, Yonsei University, Incheon 21983, Republic of KoreaMedicinal Bioconvergence Research Center, Institute for Artificial Intelligence and Biomedical Research, College of Pharmacy & College of Medicine, Gangnam Severance Hospital, Yonsei University, Incheon 21983, Republic of KoreaDivision of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea; Institute of Life Science and Natural Resources, Korea University, Seoul 02841, Republic of Korea; Corresponding authorSummary: 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).http://www.sciencedirect.com/science/article/pii/S266616672100349XProtein BiochemistryStructural BiologyX-ray Crystallography |
| spellingShingle | Sulhee Kim Ina Yoon Sunghoon Kim Kwang Yeon Hwang Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants STAR Protocols Protein Biochemistry Structural Biology X-ray Crystallography |
| 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 | Protein Biochemistry Structural Biology X-ray Crystallography |
| url | http://www.sciencedirect.com/science/article/pii/S266616672100349X |
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