Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies

Guimei Yu, Frank Vago, Dongsheng Zhang, Jonathan E. Snyder, Rui Yan, Ci Zhang, Christopher Benjamin, Xi Jiang, Richard J. Kuhn, Philip Serwer, David H. Thompson, Wen Jiang

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13 Scopus citations

Abstract

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichia coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalJournal of Structural Biology
Volume187
Issue number1
DOIs
StatePublished - Jul 2014

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Keywords

  • 3D reconstruction
  • Affinity cryo-EM
  • Affinity grid
  • Antibodies
  • Bacteriophage T7
  • Single particle cryo-EM

ASJC Scopus subject areas

  • Structural Biology

Cite this

Yu, G., Vago, F., Zhang, D., Snyder, J. E., Yan, R., Zhang, C., Benjamin, C., Jiang, X., Kuhn, R. J., Serwer, P., Thompson, D. H., & Jiang, W. (2014). Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies. Journal of Structural Biology, 187(1), 1-9. https://doi.org/10.1016/j.jsb.2014.04.006