TY - JOUR
T1 - Electron energy filtering significantly improves amplitude contrast of frozen-hydrated protein at 300 kV
AU - Yonekura, Koji
AU - Braunfeld, Michael B.
AU - Maki-Yonekura, Saori
AU - Agard, David A.
PY - 2006/12
Y1 - 2006/12
N2 - 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.
AB - 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.
KW - Amplitude contrast
KW - Cryo-electron microscopy
KW - Energy filtering
KW - Flagellar filament
UR - http://www.scopus.com/inward/record.url?scp=33750973339&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33750973339&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2006.07.016
DO - 10.1016/j.jsb.2006.07.016
M3 - Article
C2 - 16987672
AN - SCOPUS:33750973339
VL - 156
SP - 524
EP - 536
JO - Journal of Structural Biology
JF - Journal of Structural Biology
SN - 1047-8477
IS - 3
ER -