Accurate magnification calibration for transmission electron microscopy is best achieved with the use of appropriate standards and an objective calibration technique. We have developed a reliable method for calibrating the magnification of images from frozen-hydrated specimens. Invariant features in radial density plots of a standard are compared with the corresponding features in a "defocused" X-ray model of the same standard. Defocused X-ray models were generated to mimic the conditions of cryo-electron microscopy. The technique is demonstrated with polyoma virus, which was used as an internal standard to calibrate micrographs of bovine papilloma virus type 1 and bacteriophage ΦX174. Calibrations of the micrographs were estimated to be accurate to 0.35%-0.5%. Accurate scaling of a three-dimensional structure allows additional calibrations to be made with radial density plots computed from two-or three-dimensional data.

Accurate magnification calibration for transmission electron microscopy is best achieved with appropriate external or internal standards. The use of the icosahedral polyoma virus as a standard in frozen-hydrated preparations was previously described. This method used the known diameter of polyoma (from x-ray measurements) as a reference to calibrate the diameters of other viruses. Measurements were made from circular averages computed from individual particle images or from an average image of many individual particle images. The measured diameters of several different spherical viruses are in good agreement with measurements of the same viruses made with x-ray crystallographic and solution scattering techniques.We found, however, that it is very difficult to accurately measure particle diameters in images taken from frozen-hydrated specimens. This difficulty is attributable to several factors including i) uncertainty about the solvent density level, ii) superposition of density features that occur in two-dimensional projections of three-dimensional objects, iii) uneven outer surfaces of virus particles, iv) characteristically high noise and low contrast levels in micrographs, and v) contrast transfer function (CTF) effects.

Bacteriophage Φ29 is a complex-shaped, double-stranded DNA virus that infects Bacillus subtilis . Mature Φ29 particles contain six major structural proteins which give rise to several distinctive features: a prolate-shaped head (gp8), fibers (gp8.5) which radiate from the head, a connector-collar region (gp10 and gp11), collar appendages (gp12*) and a tail (gp9). Although much is known about Φ29 biochemistry, genetics and assembly, relatively little is known about its detailed structure. Three-dimensional reconstructions of the collar demonstrated that it contains two structurally distinct features: an upper portion (proximal to the head) with 12-fold axial symmetry (gp10) and a lower protion with 6-fold symmetry (possibly both gp10 and gp11). The capsid is believed to be organized with two apical regions with trimeric aggregates of gp8 dimers arranged on a T=l icosahedral lattice separated by 10 additional trimers in the equatorial region. Φ29 proheads are assembly intermediates that contain only gp8, gp8.5, gp10 and the scaffolding protein (gp7) which is released upon DNA encapsidation. We have imaged Φ29 with conventional negative-staining and the recently developed cryo-microscopy techniques with the goal of examining the structural basis for Φ29 morphogenesis.Vitrified Φ29 proheads display distinct outlines with one end pointed and the other somewhat flattened and they often orient with their long axes in the plane of the vitreous water (Fig. 1). Occasional end-on views with a circular profile are also observed. Proheads show a tendency to associate in pairs with the closest contact between the flattened ends (Fig. 1, arrow-head). Longer morphological variants are also occassionally observed (Fig. 1, arrow). The morphology of proheads is dramatically distorted in negative-stain preparations indicating significantly poorer preservation using this technique (Fig. 2). Proheads are also permeable to stain.