TY - JOUR
T1 - Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies
AU - Yu, Guimei
AU - Vago, Frank
AU - Zhang, Dongsheng
AU - Snyder, Jonathan E.
AU - Yan, Rui
AU - Zhang, Ci
AU - Benjamin, Christopher
AU - Jiang, Xi
AU - Kuhn, Richard J.
AU - Serwer, Philip
AU - Thompson, David H.
AU - Jiang, Wen
N1 - Funding Information:
This work was supported in part by NIH Grant R01AI072035 . The negative staining TEM and cryo-EM images were taken at the Purdue Biological Electron Microscopy Facility. We thank Melissa Mikolaj and Thomas J. Edwards for providing some of the Sindbis virus test samples.
PY - 2014/7
Y1 - 2014/7
N2 - 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.
AB - 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.
KW - 3D reconstruction
KW - Affinity cryo-EM
KW - Affinity grid
KW - Antibodies
KW - Bacteriophage T7
KW - Single particle cryo-EM
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U2 - 10.1016/j.jsb.2014.04.006
DO - 10.1016/j.jsb.2014.04.006
M3 - Article
C2 - 24780590
AN - SCOPUS:84902155565
SN - 1047-8477
VL - 187
SP - 1
EP - 9
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 1
ER -