Purification and cryo-EM structure determination of Arabidopsis thaliana GLR3.4

• Published in: STAR protocols 2021-12, Vol.2 (4), p.100855, Article 100855
• Main Authors: Gangwar, Shanti Pal, Green, Marriah N., Yelshanskaya, Maria V., Sobolevsky, Alexander I.
• Format: Article
• Language: English
• Subjects: Animals; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Cryo-EM; Cryoelectron Microscopy; Mammals - metabolism; Model Organisms; Plant sciences; Protein Biochemistry; Protein expression and purification; Protocol; Receptors, Glutamate - genetics
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100855

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that play crucial roles in the central nervous system. iGluR homologs, termed glutamate receptor-like channels (GLRs), have been found in plants. Investigating the structural and functional relationship between iGluRs and GLRs was limited by GLR protein expression, purification, and structural characterization. Here, we provide a detailed protocol for Arabidopsis thaliana GLR3.4 (AtGLR3.4) expression in a mammalian cell line and purification for structure determination by cryogenic electron microscopy (cryo-EM). For the complete details on the use and execution of this protocol, please refer to Green et al. (2021). [Display omitted] We provide a detailed protocol for expression and purification of Arabidopsis thaliana GLR3.4 protein that can be used for structure determination by cryo-EM. The image has been created using BioRender (https://biorender.com/). •Protocol for expression of plant glutamate receptor-like channel (GLR) in HEK cells•Purification protocol that yields pure monodisperse tetrameric GLR protein•Purified protein subjected to cryo-EM analysis produces high-resolution GLR structure Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that play crucial roles in the central nervous system. iGluR homologs, termed glutamate receptor-like channels (GLRs), have been found in plants. Investigating the structural and functional relationship between iGluRs and GLRs was limited by GLR protein expression, purification, and structural characterization. Here, we provide a detailed protocol for Arabidopsis thaliana GLR3.4 (AtGLR3.4) expression in a mammalian cell line and purification for structure determination by cryogenic electron microscopy (cryo-EM).