Cryo-EM studies of NAIP-NLRC4 inflammasomes

Methods Enzymol. 2019:625:177-204. doi: 10.1016/bs.mie.2019.04.030. Epub 2019 May 24.

Abstract

The NAIP-NLRC4 family of inflammasomes are components of the innate immune system that sound a molecular alarm in the presence of intracellular pathogens. In this chapter, we provide an in-depth guide to using cryo-electron microscopy (cryo-EM) to investigate these inflammasomes, focusing especially on the techniques we used in our recent structural analysis of the NAIP5-NLRC4 inflammasome. We explain how to circumvent specific obstacles we encountered at each step, from sample preparation through data processing. The methods described here will be useful for further studies of the NAIP5-NLRC4 inflammasome and related supracomplexes involved in innate immune surveillance; they may also be useful for unrelated complexes that present similar issues, such as preferential orientations and compositional heterogeneity.

Keywords: Cryo-EM; Flagellin; Immunology; Inflammasome; NAIP5; NLRC4; Structural biology.

Publication types

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

MeSH terms

  • Animals
  • Calcium-Binding Proteins / metabolism*
  • Calcium-Binding Proteins / ultrastructure*
  • Cryoelectron Microscopy / methods*
  • Humans
  • Immunity, Innate / physiology
  • Inflammasomes / metabolism*
  • Inflammasomes / ultrastructure*
  • Neuronal Apoptosis-Inhibitory Protein / metabolism*
  • Neuronal Apoptosis-Inhibitory Protein / ultrastructure*

Substances

  • Calcium-Binding Proteins
  • Inflammasomes
  • Neuronal Apoptosis-Inhibitory Protein