Electron energy filtering significantly improves amplitude contrast of frozen-hydrated protein at 300 kV

Koji Yonekura, Michael B. Braunfeld, Saori Maki-Yonekura, David A. Agard

Research output: Contribution to journalArticlepeer-review

40 Citations (Scopus)

Abstract

The amplitude contrast of frozen-hydrated biological samples was measured using the bacterial flagellar filament embedded in vitreous ice at an accelerating voltage of 300 kV. From the mean radial amplitude spectra of overfocused images, amplitude contrast was estimated to be 6.9 ± 1.9% and 2.7 ± 1.0% of the whole contrast at the low spatial frequency range with and without energy filtering, respectively, and that of the carbon film to be 9.5 ± 2.0% and 5.8 ± 1.8%. Energy filtering effectively doubled the signal-to-noise ratio in the images of frozen-hydrated filaments, and substantially improved intensity data statistics of layer lines up to at least ∼25 Å resolution in their Fourier transforms. It also markedly improved inter-particle fitting phase residuals of averaged data at resolutions up to ∼15 Å. Using the energy filtered data recorded on a new high-performance, lens-coupled CCD camera the three-dimensional map of the flagellar filament was calculated at 8 Å by applying the amplitude contrast of 6.9%. The map and its mean radial density distribution validated the obtained value of the amplitude contrast.

Original languageEnglish
Pages (from-to)524-536
Number of pages13
JournalJournal of Structural Biology
Volume156
Issue number3
DOIs
Publication statusPublished - 2006 Dec
Externally publishedYes

Keywords

  • Amplitude contrast
  • Cryo-electron microscopy
  • Energy filtering
  • Flagellar filament

ASJC Scopus subject areas

  • Structural Biology

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