Negative‐Stain Transmission Electron Microscopy of Molecular Complexes for Image Analysis by 2D Class Averaging
Citations Over TimeTop 10% of 2019 papers
Abstract
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.
Related Papers
- → Uranyl sulphate: A new negative stain for electron microscopy(1981)17 cited
- → Methods For Removing Uranyl Acetate Precipitate From Ultrathin Sections(1978)13 cited
- → Localization of Crystals in Diseased Oats Treated with Uranyl Acetate(1972)8 cited
- → Estimation of Single-Stage Separation Factors of Uranium Isotopes in Ion-Exchange Chromatography involving Formation of Uranyl Acetate Complexes(1982)3 cited
- → Studies on coordination compounds of uranyl acetate with some organic substances. The System: Uranyl Acetate–Pyrocatechol–Water(1963)