An optimized protocol for in vitro and in cellulo structural determination of the multi-tRNA synthetase complex by cross-linking mass spectrometry

STAR Protoc. 2022 Mar 9;3(1):101201. doi: 10.1016/j.xpro.2022.101201. eCollection 2022 Mar 18.

Abstract

Despite recent advances in structural determination of individual proteins, elucidating the 3-dimensional architecture of large, multiprotein complexes remains challenging, partly because of issues related to structural integrity during purification. Here, we describe a protocol to determine the 3-dimensional architecture of the 11-constituent, multi-tRNA synthetase complex (MSC) using chemical cross-linking coupled with mass-spectrometry (XL-MS). The protocol does not require purification and is broadly applicable, facilitating determination of native structures in cell lysates and in non-disrupted cells as well as in purified complexes. For complete details on the use and execution of this protocol, please refer to Khan et al. (2020).

Keywords: Cell Biology; Chemistry; Mass Spectrometry; Molecular Biology; Protein Biochemistry; Structural Biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acyl-tRNA Synthetases*
  • Mass Spectrometry / methods
  • Multiprotein Complexes / chemistry
  • Proteins / chemistry

Substances

  • Multiprotein Complexes
  • Proteins
  • Amino Acyl-tRNA Synthetases