Low-dose electron image reconstruction of negatively stained contractile phage sheath from Bacillus subtilis (PBS-Z)

The structure of the contractile sheath of the defective phage from B. subtilis (PBS-Z) has been investigated by low-dose electron microscopy and image reconstruction. The extended and contracted sheath particles were imaged by means of two negative stains which consisted of uranyl- and phosphotungstate-containing solutions of a pH of 4.2 and 7.0 respectively. Images of identical parts of the same type of specimen were recorded at a total electron dose of 80 C/m2 (5 electrons/A2) and 4 x 10(3) C/m2 (250 electrons/A2). The low-dose reconstructions of the extended and contracted sheath structure in the two stains show good correspondence and made it possible to draw the following structural conclusions. The sheath protein in both types of structure has an elongated shape, and in both structures the long molecular axis lies in a plane perpendicular to the helical sheath axis. The orientation of the protein in the extended and contracted sheath is different; the long axes differ by about 35 degrees in orientation. The reconstructions did not permit conclusions about different conformational states of the protein in both structures. These data, together with the packing parameters of the protein subunits in the contractile sheath [1], form the complete structural analysis of this biological structure by electron microscopy. The radiation damage effects which have been monitored in analyzing image pairs to the full extent may be summarized as follows. (1) Diameters of the sheath structure increase, which indicate flattening. (2) There is no loss in resolution, and layerline altitudes of the Fourier-transformed images do not change. (3) Uranyl stain behaves differently compared to phosphotungstate. In both negative stains the structural noise level increases upon irradiation as follows from the increase in phase residuals of the digital layerline data. In uranyl-stained images also more aperiodic noise appears. (4) The Fourier amplitudes of the principal layerline maxima shift towards lower spatial frequencies; phases of corresponding maxima generally remain constant. This pattern is more pronounced in the extended sheath data; there is no rationale describing these positional shifts. Moreover, in the case of contracted sheath the amplitudes of Fourier components also change more in absolute value. Therefore the damage effects also seem to depend on the type of structure embedded in the stain. (5) In the reconstructed images these radiation effects create artificial stain-excluded volumes of a type and at a radius which depend on the stain and structure.