Avoiding the pitfalls of single particle cryo-electron microscopy: Einstein from noise

Abstract

Single particle cryo-electron microscopy is currently poised to produce high-resolution structures of many biological assemblies, but several pitfalls can trap the unwary. This critique highlights one problem that is particularly relevant when smaller structures are being studied. It is known as "Einstein from noise," in which the experimenter honestly believes they have recorded images of their particles, whereas in reality, most if not all of their data consist of pure noise. Selection of particles using cross-correlation methods can then lead to 3D maps that resemble the model used in the initial selection and provide the illusion of progress. Suggestions are given about how to circumvent the problem.

Keywords: 3DEM; EMDB; SPEM; cryo-EM; validation.

Fig. 1.

Illustration taken from a paper describing model bias (23). The image is copied from figure 2A in that paper. The familiar photograph of Einstein emerged from 1,000 images of pure white noise, after alignment to the model using a cross-correlation function. Reprinted from Journal of Structural Biology, Vol. 166, M. Shatsky et al., A method for the alignment of heterogeneous macromolecules from electron microscopy, pp. 67–78, Copyright 2009, with permission from Elsevier.

Fig. 2.

(A–F) Six individual windowed images from the stack of 423 that was supplied by the authors (21). (G) Average of 423 windowed images using the same gray scale as A–F. (H) Average of 423 windowed images with 15× increased contrast. The density in the central region of G and H shows the average of the many views used in particle picking and verification. The circle of dark density round the edge of the average, seen more clearly in H should not be present in the raw images so must arise from masked projections from the 3D map or model used to extract the particles. (I–L) Difference maps obtained by subtraction of sections from the two independent half maps [i.e., maps calculated using only half the data, normally even and odd particles in the stack (18)] supplied by the authors (21). The four panels represent sections at different heights along the spike, viewed from the apex. The differences are confined to a sharply defined region with no gradation into the flat background. This clearly visible and relatively sharp mask serves to constrain any density to the region inside the mask during iterative refinement. Use of masking plus the same initial reference suggests how the apparent resolution was extended from 11 to 6 Å. All images are on the same scale, with a window size of 190 Å.

Fig. 3.

Cryo-EM image of a field of view of β-galactosidase single particles (molecular weight, 450 kDa). This image (number 04.51.13) was recorded on an FEI Falcon II detector at 300 keV and 80,000x magnification with a dose of 100 el/Ų and a defocus of 3.5 μm. The particles are all clearly visible at a defocus value similar to that used to obtain the images presented in figure S1A of Mao et al. (21).