The effects of radiation damage on the structure of frozen hydrated HSV-1 capsids.
Radiation damage imposes stringent limits on the information content of electron micrographs of biological specimens. In this study, we have investigated its effects on frozen, hydrated specimens and three-dimensional 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 (which are fully packaged) were recorded and reconstructions were calculated from the first exposures, corresponding to a cumulative electron dose of 6-7 e-/A2, and from later exposures (25-40 e-/A2). Experimental procedures were standardized to ensure that perceived changes in the micrographs and reconstructions would be attributable to radiation damage alone. The effects of the higher doses in both the micrographs and the reconstructions were expressed as a progressive blurring of the finer details, corresponding to a delocalization of structure in the ice-embedded specimens. The resolutions of the reconstructions were quantified according to a form of the Fourier ring correlation coefficient criterion, according to which the first-exposure reconstructions had resolutions of 30-36 A. The fifth-exposure B-capsid reconstruction had comparable nominal resolution, although it exhibited progressively lower correlations at higher spatial frequencies. Qualitatively similar changes in the series of C-capsid reconstructions were observed although they were more pronounced, presumably because these micrographs had lower contrast and signal-to-noise ratios. We infer that the observed changes in the images and reconstructions and the concomitant loss in contrast in the immediate vicinity of the capsid surface may reflect radiation-induced perturbation of molecular structure and/or the release of peptide fragments. Nevertheless, the observed changes are relatively subtle, at least at the operational resolution of this study; overall, our results support earlier indications (M. F. Schmid et al. J. Struct. Biol. 108, 62-68, 1992) that prospects are quite good for tilt-series reconstructions from cryoelectron micrographs, including six to eight views of the same specimen.
Conway
J F
JF
Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Trus
B L
BL
Booy
F P
FP
Newcomb
W W
WW
Brown
J C
JC
Steven
A C
AC
D002213:Capsid / Q000528:radiation effects* / Q000648:ultrastructure*; D004583:Electrons; D005615:Freezing; D018259:Herpesvirus 1, Human / Q000528:radiation effects* / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D014867:Water
D014867:Water