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

• DOI: 10.1016/j.jsb.2014.04.006
• Publication Date: 2014-04-26T16:18:46Z
• Authors: 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
• Keywords: Cryo-Electron Microscopy; Single-Particle Analysis; Structural Biology; Particle (ecology)

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 Escherichiacoli 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.