Use of radial density plots to calibrate image magnification for frozen-hydrated specimens

Abstract

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.

Three-dimensional structure of poliovirus receptor bound to poliovirus

“…The R-factor criterion gave a similar result. After calibrating the size and density of the virion-sPvr reconstruction to that of the virion by comparing the radially averaged density (29) in the capsid region only, a difference map was computed.…”

A Model-Based Approach for Determining Orientations of Biological Macromolecules Imaged by Cryoelectron Microscopy

“…One way to check the relative scales of the data is to compare ID radial density plots (e.g., Olson and Baker, 1989;Belnap et al, 1993) of the circularly symmetrized reference projection (Fig. 2) and a circularly symmetrized, global average of the images.…”

Conserved Features in Papillomavirus and Polyomavirus Capsids

“…A preliminary 3D reconstruction of CRPV was obtained by means of established crosscorrelation (Belnap et al, 1993;Baker et al, 1990) and common-lines procedures (Fuller, 1987;Baker et al, 1988;Fuller et al, 1996). Then, a model-based, polar-Fourier-transform (PFT) method (Cheng et al, 1994; was used to determine initial orientation (Θ,Φ,Ω) and translation (x,y origin) parameters for all the BPV-1, CRPV, HPV-1, HPV-1 VLP, and SV40 particle images.…”

“…They also appear on the 3-fold axes where they are bounded by d and e subunits. a, Adolph et al (1979); b, Anderer et al (1967); c, Baker et al (1983); d, Baker et al (1988Baker et al ( , 1989); e, Baker et al (1991); f, Belnap et al (1993), Baker (1992); g, Caspar (1966); h, Crowther & Amos (1972); i, Crowther et al (1970a);j, Finch (1974); k, ; l, Griffith et al (1992); m, Hagensee et al (1994); n, Kiselev & Klug (1969); o, Klug (1965); p, ; q, Klug & Finch (1968); r, Liddington et al (1991); s, Rayment et al (1982); t, Salunke et al (1986);u, Stehle et al (1994); v, this study; w, Yabe et al (1979). are averages for each set of particle images and were computed over the full particle radius.…”