Abstract

Andrea Fera37007*

Electron microscopy tomography results obtained after increasing the resistance of flash-frozen proteins to accelerated electron beams. This curing reduces electron dose dependence, allowing high-resolution tomography without need of averaging multiple samples. Three-dimensional images at atomic-scale are realized on one individual protein in one experiment. This workflow, introduced for flash-frozen rigid biopolymers, is here generalized and applied to flash-frozen, deformable oligomers extracted from a virus. Results are shown down to atomic coordinates without recurring to averaging among many samples. It is possible, therefore, to measure unique, individual enzymes during interactions with proteins. In fact, results like this are usual when imaging by electron microscopy semiconductors or other solid materials, because such samples are not damaged by interactions with accelerated electron beams in vacuum. Data obtained applying this workflow may allow to speed-up understanding of protein-protein interactions, leading to focus on effective drugs. A rationale of this behavior is proposed, stemming out of the discrete nature of interactions between high-energy electron beams and matter. Finally, this workflow offers the new possibility to store samples under liquid nitrogen after the first observation, for more imaging of the same area in more details; a characteristic useful for imaging large protein groups.

Here we document results obtained by electron microscopy tomography after increasing the resistance of flash-frozen viral proteins to accelerated electron beams, consequent to a few hours’ treatment in high vacuum at cryo-temperatures. Such temperature curing greatly reduces dose limitations, therefore allowing high-resolution tomography experiments also on flash frozen protein aggregates, and without the need of averaging multiple samples. In this way three-dimensional atomic scale images can be realized on one individual aggregate and in one experiment. This workflow has been tentatively introduced for flash-frozen rigid biopolymers, but only here this method is further generalized and applied to flash-frozen, deformable oligomers of proteins extracted from the HIV-1 virus. These results indicate that protein constructs can be imaged down to atomic coordinates by electron microscopy tomography without recurring to averaging among many samples. It will be possible, therefore, to measure unique, individual molecules also during the timed interaction with other proteins. In fact, results at this scale are usual with electron microscopy when imaging semiconductors or other solid materials, because such samples are not quickly damaged by interactions with accelerated electron beams in vacuum. In summary, data obtained applying this workflow could allow to speed-up significantly the understanding of protein- protein interactions, leading biological research to focus on effective drugs. A tentative rationale of this behavior is proposed, stemming out of the discreet nature of the interactions between high-energy electron beams and matter.

Finally, this workflow offers the possibility, new to cryo-electron microscopy of biologic samples, to store a sample indefinitely under liquid nitrogen after the first observation, for more imaging of the same area in more details. This characteristic may be useful for imaging complex tissues, or large groups of many proteins down to atomic resolution, like e.g. in brain tissue.