Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.
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.
Markey Center for Structural Biology, Department of Biological Science, Purdue University, West Lafayette, IN, USA.
Guimei
G
Yu
Markey Center for Structural Biology, Department of Biological Science, Purdue University, West Lafayette, IN, USA.
Frank
F
Vago
Divisions of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Dongsheng
D
Zhang
Markey Center for Structural Biology, Department of Biological Science, Purdue University, West Lafayette, IN, USA.
Jonathan E
JE
Snyder
Markey Center for Structural Biology, Department of Biological Science, Purdue University, West Lafayette, IN, USA.
Rui
R
Yan
Markey Center for Structural Biology, Department of Biological Science, Purdue University, West Lafayette, IN, USA.
Ci
C
Zhang
Department of Chemistry, Purdue University, West Lafayette, IN, USA.
Christopher
C
Benjamin
Divisions of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Xi
X
Jiang
Markey Center for Structural Biology, Department of Biological Science, Purdue University, West Lafayette, IN, USA.
Richard J
RJ
Kuhn
Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Philip
P
Serwer
Department of Chemistry, Purdue University, West Lafayette, IN, USA.
David H
DH
Thompson
Markey Center for Structural Biology, Department of Biological Science, Purdue University, West Lafayette, IN, USA. Electronic address: jiang12@purdue.edu.
Wen
W
Jiang
D000906:Antibodies / Q000737:chemistry; D000915:Antibody Affinity; D017123:Bacteriophage T7 / Q000648:ultrastructure*; D002139:Caliciviridae / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D054681:Ribosome Subunits, Large, Bacterial / Q000648:ultrastructure*; D012845:Sindbis Virus / Q000648:ultrastructure*; D013205:Staphylococcal Protein A / Q000737:chemistry
D000906:Antibodies; D013205:Staphylococcal Protein A
3D reconstruction; Affinity cryo-EM; Affinity grid; Antibodies; Bacteriophage T7; Single particle cryo-EM