Inactivation of catalase monolayers by irradiation with 100 keV electrons.

Abstract

A catalase monolayer adsorbed on a layer of arachidic acid deposited on a solid support was irradiated with 100 keV electrons simulating the conditions of electron microscopic imaging. Effective doses were calculated taking into account the angular and energy distribution of backscattered electrons. Enzymatic inactivation was chosen as the criterion for damage and was monitored by a rapid and quantifiable but nevertheless sensitive assay. Dose-response curves revealed that inactivation is a one-hit-multiple-target phenomenon, which is consistent with biochemical evidence for a cooperative function of subunits. The experimentally determined target size coincides fairly well with both calculated cross sections for inelastic interactions based on the atomic composition of catalase and with calculated cross sections for ionizing events based on the chemical bonds involved. This legitimates both types of calculations even for complex biomolecules. Macromolecules and supramolecular assemblies are subject to substantial structural alterations when exposed to the unfavorable conditions in the electron microscope. Theoretical considerations (1) as well as experimental investigations (for review see ref.2) led to the suspicion that radiation-induced deteriorations of the molecular structure might fundamentally limit the resolution to which reliable structural information can be obtained. The degree of structural alteration is a function of the radiation sensitivity of the specimen under investigation and of the electron dose deposited on it, which in turn is defined by the wanted resolution (3, 4). Subtle structural alterations which can be neglected at moderate resolution levels become increasingly important with increasing resolution.The complexity of the phenomena associated with radiation damage of proteins or biomolecules in general made it impossible to formulate inductively a universal theory of the damaging process (for reviews see refs. 5-7) which could define or even predict the kind and degree of structural alterations following the fairly well understood physical interaction between the beam electrons and the specimen atoms. For essentially the same reason it is impossible to trace back on a theoretical basis to the original undisturbed structure of a protein from micrographs with manifest deteriorations.To evaluate realistic perspectives for "molecular microscopy" it seems necessary to measure quantitatively the electron doses causing distinct structural deterioration in various molecular species of biological relevance and under experimental conditions simulating the hazards of electron microscopical imaging. Hitherto, radiation damage was often measured by determining the dose that caused fading of distinct spots of the electron diffraction pattern. Unfortunately, this method is confined to crystalline specimens and, moreover, the situation of a molecule in a crystal need not necessarily reflect its reaction to ionizing radiation in a noncrystalline state. Organized monolayer systems assembled in a ...

The role of electron irradiation history in liquid cell transmission electron microscopy

“…The cryogenic transmission electron microscopy (cryo-EM) field has shown a tolerable structural threshold of 5 to 20 e − /Å 2 cumulatively for macromolecular complexes ( 33 ) and 50 to 200 e − /Å 2 for whole cells depending on target resolution ( 34 ). However, although structural information may be intact at these electron fluxes, enzyme activity has been demonstrated to be altered with fluxes in the range of 0.05 e − /Å 2 ( 35 ). To date, a rigorous investigation of the cumulative irradiation sensitivity to biological samples in the confined environment of thin liquid cells used for LC-TEM has not been performed, although several studies have attempted to demonstrate the viability of cells irradiated with the electron beam by using so-called live/dead cell fluorescent assays ( 3 , 36 ).…”

Specificity of stain distribution in electron micrographs of protein molecules contrasted with uranyl acetate

“…It should be recalled, however, that the net agreement between our experimental images and the corresponding simulations is remarkably good. That this should be so is surprising, in view of the extreme effects which electron irradiation, in particular, has been shown to exert on the structural integrity (Isaacson, 1977) and functional activity (Hahn et al, 1976) of proteins. To rationalize this observation, we conjecture that negative stain, in addition to providing contrast, may preserve macromolecular structure (Kellenberger & Kistler, 1978), or at least a record of structure in terms of the relatively insensitive distribution of heavy atoms (Baumeister & Hahn, 1975;Hoppe et al, 1975).…”

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

“…Despite this knowledge it remains unknown how a loss of structure impacts the functionality of biomolecules, whether the catalytic mechanism of an enzyme, the protein coding function of an mRNA, or the transport, recognition, and binding of a transcription factor. Dehydrated catalase proteins have been demonstrated to lose their catalytic function at electron flux rates as low as 0.05 e -/Å 2 (32). However, it remains unclear how similar a hydrated protein sample may behave in LC-TEM due to the added secondary damage mechanisms from radiolysis.…”