Open AccessNegative‐Stain Transmission Electron Microscopy of Molecular Complexes for Image Analysis by 2D Class Averaging
Author(s) -
Gallagher John R.,
Kim Alexander J.,
Gulati Neetu M.,
Harris Audray K.
Publication year - 2019
Publication title -
current protocols in microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.813
H-Index - 35
eISSN - 1934-8533
pISSN - 1934-8525
DOI - 10.1002/cpmc.90
Subject(s) - uranyl acetate , negative stain , resolution (logic) , stain , hemagglutinin (influenza) , transmission electron microscopy , uranyl , epitope , image (mathematics) , microscopy , domain (mathematical analysis) , electron microscope , biology , crystallography , biophysics , physics , chemistry , optics , computer science , artificial intelligence , staining , virology , mathematics , genetics , mathematical analysis , uranium , nuclear physics , virus , antigen
Negative‐stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo‐EM image processing have maximized the information gained from averaging large numbers of particles. These developments can now be applied back to negative‐stain image analysis to ascertain domain level molecular structure (10 to 20 Å) more quickly and efficiently than possible by atomic resolution cryo‐EM. Using uranyl acetate stained molecular complexes of influenza hemagglutinin bound to Fab 441D6, we describe a simple and efficient means to collect several hundred micrographs with SerialEM. Using RELION, we illustrate how tens of thousands of complexes can be auto‐picked and classified to accurately describe the domain level topology of this unconventional hemagglutinin head‐domain epitope. By comparing to the cryo‐EM density map of the same complex, we show that questions about epitope mapping and conformational heterogeneity can readily be answered by this negative‐stain method. © 2019 The Authors.