Quantitative STEM mass measurement of biological macromolecules in a 300 kV TEM.
For almost four decades, the scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold field-emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can also provide accurate STEM mass measurements. Using the recently published database of elastic-scattering cross sections from the National Institute of Standards and Technology, we show that the measured absolute mass values for tobacco mosaic virus and limpet hemocyanin didecamers agree with the known values to within better than 10%. Applying the established approach, whereby tobacco mosaic virus is added to a specimen as a calibration standard, we find that the measured molecular weight of the hemocyanin assemblies agrees with the known value to within 3%. This accuracy is achievable although only a very small fraction ( approximately 0.002) of the incident probe current of 300 kV electrons is scattered onto the annular dark-field STEM detector. FEG TEMs operating at intermediate voltages (200-400 kV) are becoming common tools for determining the structure of frozen hydrated protein assemblies. The ability to perform mass determination with the same instrument can provide important complementary information about the numbers of subunits comprising the protein assemblies whose structure is being studied.
Sousa
Alioscka A
AA
National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD 20892, USA.
Leapman
Richard D
RD
D006433:Hemocyanins / Q000737:chemistry; D046911:Macromolecular Substances / Q000737:chemistry*; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D008970:Molecular Weight; D014027:Tobacco Mosaic Virus / Q000737:chemistry
D046911:Macromolecular Substances; D006433:Hemocyanins