THE EFFECTS OF RADIATION-DAMAGE ON THE STRUCTURE OF FROZEN-HYDRATED HSV-1 CAPSIDS

Radiation damage imposes stringent limits on the information content o f electron micrographs of biological specimens. In this study, we have investigated its effects on frozen, hydrated specimens and three-dime nsional reconstructions calculated from cryomicrographs using capsids of herpes simplex virus as a model system. Multiple-exposure series of micrographs of both B-capsids (which contain no DNA) and C-capsids (w hich are fully packaged) were recorded and reconstructions were calcul ated from the first exposures, corresponding to a cumulative electron dose of 6-7 e(-)/Angstrom(2), and from later exposures (25-40 e(-)/Ang strom(2)). Experimental procedures were standardized to ensure that pe rceived changes in the micrographs and reconstructions would be attrib utable to radiation damage alone. The effects of the higher doses in b oth the micrographs and the reconstructions were expressed as a progre ssive blurring of the finer details, corresponding to a delocalization of structure in the ice-embedded specimens. The resolutions of the re constructions were quantified according to a form of the Fourier ring correlation coefficient criterion, according to which the first-exposu re reconstructions had resolutions of 30-36 Angstrom. The fifth-exposu re B-capsid reconstruction had comparable nominal resolution, although it exhibited progressively lower correlations at higher spatial frequ encies. Qualitatively similar changes in the series of C-capsid recons tructions were observed although they were more pronounced, presumably because these micrographs had lower contrast and signal-to-noise rati os. We infer that the observed changes in the images and reconstructio ns and the concomitant loss in contrast in the immediate vicinity of t he capsid surface may reflect radiation-induced perturbation of molecu lar structure and/or the release of peptide fragments. Nevertheless, t he observed changes are relatively subtle, at least at the operational resolution of this study; overall, our results support earlier indica tions (M. F. Schmid et al. J. Struct. Biol. 108, 62-68, 1992) that pro spects are quite good for tilt-series reconstructions from cryoelectro n micrographs, including six to eight views of the same specimen. (C) 1993 Academic Press, Inc.