A step-by-step protocol for capturing conformational snapshots of ligand gated ion channels by single-particle cryo-EM

Kaihua Zhang; David Julius; Yifan Cheng

doi:10.1016/j.xpro.2022.101732

A step-by-step protocol for capturing conformational snapshots of ligand gated ion channels by single-particle cryo-EM

STAR Protoc. 2022 Dec 16;3(4):101732. doi: 10.1016/j.xpro.2022.101732. Epub 2022 Sep 30.

Authors

Kaihua Zhang¹, David Julius², Yifan Cheng³

Affiliations

¹ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
² Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: david.julius@ucsf.edu.
³ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: yifan.cheng@ucsf.edu.

Abstract

Capturing conformational snapshots by single-particle cryo-EM facilitates the analysis of ligand binding and activation mechanisms for ion channels and other receptor complexes. Here, we present a protocol to capture intermediate states of nanodisc-reconstituted TRPV1. This protocol covers sample preparation, data acquisition, and image processing with focuses on the symmetry expansion and focused 3D classification. This protocol can be adapted to different proteins and samples. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Keywords: Cryo-EM; Structural biology.

Publication types

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

MeSH terms

Cryoelectron Microscopy / methods
Image Processing, Computer-Assisted / methods
Ligand-Gated Ion Channels*
Molecular Conformation
Proteins

Substances

Ligand-Gated Ion Channels
Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding