Preparation of filamentous proteins for electron microscopy visualization and reconstruction

Mariusz Matyszewski, Jungsan Sohn

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Cryo electron microscopy (cryo-EM) has become a mainstream tool for determining the structures of macromolecular complexes at the atomic resolution. It has many advantages over other techniques such as X-ray crystallography and nuclear magnetic resonance (NMR). However, it also entails several challenges, a major one being preparation of an ideal sample. Recent studies have identified that DNA sensors and inflammasomes often assemble into filamentous oligomers, which poses a unique set of challenges in preparing ideal samples for high-resolution reconstruction using cryo-EM. This chapter will discuss how to overcome several major issues in cryo-EM sample preparation including construct design, screening using negative stain (ns) EM, and tips on working with filamentous proteins.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
Publication statusPublished - Jan 1 2019

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

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Keywords

  • Cryo-EM
  • Filaments
  • Fusion tags
  • Negative stain EM
  • Sample preparation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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