Native Mass Spectrometry-Based Screening for Optimal Sample Preparation in Single-Particle Cryo-EM

Structure. 2021 Feb 4;29(2):186-195.e6. doi: 10.1016/j.str.2020.11.001. Epub 2020 Nov 19.


Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.

Keywords: SARS-CoV-2 replication-transcription complex; bacterial RNA polymerase; biochemical screening; cryo-EM; cryo-EM sample quality check; integrated structural biology; native mass spectrometry; protein complexes; transcription.

Publication types

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

MeSH terms

  • Cryoelectron Microscopy / methods*
  • Escherichia coli
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Mass Spectrometry / methods*
  • Methyltransferases / chemistry
  • Methyltransferases / metabolism
  • RNA Helicases / chemistry
  • RNA Helicases / metabolism
  • SARS-CoV-2 / enzymology
  • SARS-CoV-2 / ultrastructure
  • Single Molecule Imaging / methods*
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / metabolism


  • Escherichia coli Proteins
  • TraR protein, E coli
  • Transcription Factors
  • Viral Nonstructural Proteins
  • Methyltransferases
  • Nsp13 protein, SARS-CoV
  • RNA Helicases