Membrane Protein Cryo-EM: Cryo-Grid Optimization and Data Collection with Protein in Detergent

Methods Mol Biol. 2020;2127:227-244. doi: 10.1007/978-1-0716-0373-4_16.


Cryo-electron microscopy (cryo-EM) is a powerful tool for investigating the structure of macromolecules under near-native conditions. Especially in the context of membrane proteins, this technique has allowed researchers to obtain structural information at a previously unattainable level of detail. Specimen preparation remains the bottleneck of most cryo-EM research projects, with membrane proteins representing particularly challenging targets of investigation due to their universal requirement for detergents or other solubilizing agents. Here we describe preparation of negative staining and cryo-EM grids and downstream data collection of membrane proteins in detergent, by far the most common solubilization agent. This protocol outlines a quick and straightforward procedure for screening and determining the structure of a membrane protein of interest under biologically relevant conditions.

Keywords: Cryo-electron microscopy; Data collection; Detergent; Grid preparation; Membrane protein; Negative staining; Structural biology; Vitrification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calibration
  • Computer Systems / standards
  • Cryoelectron Microscopy / instrumentation
  • Cryoelectron Microscopy / methods*
  • Cryoelectron Microscopy / standards
  • Data Collection / methods*
  • Data Collection / standards
  • Detergents / chemistry
  • Detergents / pharmacology*
  • Humans
  • Membrane Proteins / chemistry*
  • Membrane Proteins / drug effects
  • Membrane Proteins / isolation & purification
  • Microscopy, Electron, Transmission / instrumentation
  • Microscopy, Electron, Transmission / methods
  • Microscopy, Electron, Transmission / standards
  • Negative Staining / instrumentation
  • Negative Staining / methods
  • Negative Staining / standards
  • Protein Denaturation / drug effects
  • Specimen Handling / instrumentation
  • Specimen Handling / methods


  • Detergents
  • Membrane Proteins