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

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

STAR protocols (2021-10-15)
Shanti Pal Gangwar, Marriah N Green, Maria V Yelshanskaya, Alexander I Sobolevsky
ABSTRACT

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).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Pepstatin A, microbial, ≥75% (HPLC)
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Leupeptin hydrochloride, microbial, ≥70% (HPLC)
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d-Desthiobiotin, ≥98% (TLC)
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Aprotinin from bovine lung, lyophilized powder, 3-8 TIU/mg solid
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Gentamicin sulfate, meets USP testing specifications, powder
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L-Glutamic acid monosodium salt monohydrate, ≥98.0% (NT)