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J Struct Biol. 2011 Sep;175(3):253-63. doi: 10.1016/j.jsb.2011.05.004. Epub 2011 May 24.

Limiting factors in atomic resolution cryo electron microscopy: no simple tricks.

Author information

1
Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, 237 BSRB, 615 Charles E. Young Dr. S., Los Angeles, CA 90095-7364, USA.

Abstract

To bring cryo electron microscopy (cryoEM) of large biological complexes to atomic resolution, several factors--in both cryoEM image acquisition and 3D reconstruction--that may be neglected at low resolution become significantly limiting. Here we present thorough analyses of four limiting factors: (a) electron-beam tilt, (b) inaccurate determination of defocus values, (c) focus gradient through particles, and (d) particularly for large particles, dynamic (multiple) scattering of electrons. We also propose strategies to cope with these factors: (a) the divergence and direction tilt components of electron-beam tilt could be reduced by maintaining parallel illumination and by using a coma-free alignment procedure, respectively. Moreover, the effect of all beam tilt components, including spiral tilt, could be eliminated by use of a spherical aberration corrector. (b) More accurate measurement of defocus value could be obtained by imaging areas adjacent to the target area at high electron dose and by measuring the image shift induced by tilting the electron beam. (c) Each known Fourier coefficient in the Fourier transform of a cryoEM image is the sum of two Fourier coefficients of the 3D structure, one on each of two curved 'characteristic surfaces' in 3D Fourier space. We describe a simple model-based iterative method that could recover these two Fourier coefficients on the two characteristic surfaces. (d) The effect of dynamic scattering could be corrected by deconvolution of a transfer function. These analyses and our proposed strategies offer useful guidance for future experimental designs targeting atomic resolution cryoEM reconstruction.

PMID:
21627992
PMCID:
PMC3710782
DOI:
10.1016/j.jsb.2011.05.004
[Indexed for MEDLINE]
Free PMC Article

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