Cryo-electron tomography related radiation-damage parameters for individual-molecule 3D structure determination

Abstract

To understand the dynamic structure–function relationship of soft- and biomolecules, the determination of the three-dimensional (3D) structure of each individual molecule (nonaveraged structure) in its native state is sought-after. Cryo-electron tomography (cryo-ET) is a unique tool for imaging an individual object from a series of tilted views. However, due to radiation damage from the incident electron beam, the tolerable electron dose limits image contrast and the signal-to-noise ratio (SNR) of the data, preventing the 3D structure determination of individual molecules, especially at high-resolution. Although recently developed technologies and techniques, such as the direct electron detector, phase plate, and computational algorithms, can partially improve image contrast/SNR at the same electron dose, the high-resolution structure, such as tertiary structure of individual molecules, has not yet been resolved. Here, we review the cryo-electron microscopy (cryo-EM) and cryo-ET experimental parameters to discuss how these parameters affect the extent of radiation damage. This discussion can guide us in optimizing the experimental strategy to increase the imaging dose or improve image SNR without increasing the radiation damage. With a higher dose, a higher image contrast/SNR can be achieved, which is crucial for individual-molecule 3D structure. With 3D structures determined from an ensemble of individual molecules in different conformations, the molecular mechanism through their biochemical reactions, such as self-folding or synthesis, can be elucidated in a straightforward manner.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes

“…Third, although the role ice plays in radiation damage is not currently well understood, it is possible that embedding the particles in the ice present in cryo-landed samples leads to less radiation damage by the electron beam. 20 Finally, in the case of β-galactosidase, we observe more orientations in the cryolanded data shown here, presumably due to the reduced particle−carbon surface interactions. Despite these considerable improvements over room temperature landing, we have not yet achieved structural resolution comparable to that of conventional plunge-frozen cryo-EM samples.…”

“…Second, the thin film of ice could partially play the protective role of the chemical matrix we recently described. Third, although the role ice plays in radiation damage is not currently well understood, it is possible that embedding the particles in the ice present in cryo-landed samples leads to less radiation damage by the electron beam . Finally, in the case of β-galactosidase, we observe more orientations in the cryo-landed data shown here, presumably due to the reduced particle–carbon surface interactions.…”

Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature

“…3), the GroEL molecules were barely distinguishable from the background. This dose is about 10× less than what is generally acceptable for vitrified protein samples in cryo-EM 28 . This shortcoming is expected for room temperature imaging as low temperature is known to slow down the processes induced by the electron beam 28 .…”

High-Resolution Electron Diffraction of Hydrated Protein Crystals at Room Temperature

“…Since the effect of radiation exposure on a crystal depends on many factors (crystal thickness, volume of surrounding liquid, etc. ), it is difficult to predict the maximum e-dose that a crystal can tolerate . To circumvent the a priori unknown dose limit, the integration mode of the hybrid-pixel detector was used in the data collection (see Methods section).…”