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Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging.

Zanetti G, Riches JD, Fuller SD, Briggs JA - J. Struct. Biol. (2009)

Bottom Line: The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF.Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination.We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRD1 as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.

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Effect of correction on averaged PRD1 sub-tomograms. (A) Fourier shell correlation between averaged maps obtained from aligned sub-tomograms split in two datasets. The 0.5 and the 3σ threshold curves are plotted for comparison. Blue line: uncorrected, the resolution is 29 Å at 3σ. Red line: corrected using the mean defocus value for all images, the resolution is 22 Å at 3σ. Orange line: corrected using the defocus value calculated from the relative magnification, the resolution is 22 Å at 3σ. (B) FSC between averaged maps obtained from aligned sub-tomograms and an appropriately scaled and aligned electron density map obtained from the available atomic model (PDB 1W8X). The colour code corresponds to that in (A). (C) Portion of the 3D map obtained from uncorrected sub-tomogram averaging. (D) Corresponding portion of the 3D map obtained from averaging of sub-tomograms after correction based on the mean defocus value. Scale bar is 100 Å.
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fig5: Effect of correction on averaged PRD1 sub-tomograms. (A) Fourier shell correlation between averaged maps obtained from aligned sub-tomograms split in two datasets. The 0.5 and the 3σ threshold curves are plotted for comparison. Blue line: uncorrected, the resolution is 29 Å at 3σ. Red line: corrected using the mean defocus value for all images, the resolution is 22 Å at 3σ. Orange line: corrected using the defocus value calculated from the relative magnification, the resolution is 22 Å at 3σ. (B) FSC between averaged maps obtained from aligned sub-tomograms and an appropriately scaled and aligned electron density map obtained from the available atomic model (PDB 1W8X). The colour code corresponds to that in (A). (C) Portion of the 3D map obtained from uncorrected sub-tomogram averaging. (D) Corresponding portion of the 3D map obtained from averaging of sub-tomograms after correction based on the mean defocus value. Scale bar is 100 Å.

Mentions: Using this approach, a map of PRD1 was obtained from uncorrected images with a resolution of 30 Å at the 3σ threshold and of 31 Å at the 0.5 threshold of the FSC (Fig. 5A, blue line).


Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging.

Zanetti G, Riches JD, Fuller SD, Briggs JA - J. Struct. Biol. (2009)

Effect of correction on averaged PRD1 sub-tomograms. (A) Fourier shell correlation between averaged maps obtained from aligned sub-tomograms split in two datasets. The 0.5 and the 3σ threshold curves are plotted for comparison. Blue line: uncorrected, the resolution is 29 Å at 3σ. Red line: corrected using the mean defocus value for all images, the resolution is 22 Å at 3σ. Orange line: corrected using the defocus value calculated from the relative magnification, the resolution is 22 Å at 3σ. (B) FSC between averaged maps obtained from aligned sub-tomograms and an appropriately scaled and aligned electron density map obtained from the available atomic model (PDB 1W8X). The colour code corresponds to that in (A). (C) Portion of the 3D map obtained from uncorrected sub-tomogram averaging. (D) Corresponding portion of the 3D map obtained from averaging of sub-tomograms after correction based on the mean defocus value. Scale bar is 100 Å.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806944&req=5

fig5: Effect of correction on averaged PRD1 sub-tomograms. (A) Fourier shell correlation between averaged maps obtained from aligned sub-tomograms split in two datasets. The 0.5 and the 3σ threshold curves are plotted for comparison. Blue line: uncorrected, the resolution is 29 Å at 3σ. Red line: corrected using the mean defocus value for all images, the resolution is 22 Å at 3σ. Orange line: corrected using the defocus value calculated from the relative magnification, the resolution is 22 Å at 3σ. (B) FSC between averaged maps obtained from aligned sub-tomograms and an appropriately scaled and aligned electron density map obtained from the available atomic model (PDB 1W8X). The colour code corresponds to that in (A). (C) Portion of the 3D map obtained from uncorrected sub-tomogram averaging. (D) Corresponding portion of the 3D map obtained from averaging of sub-tomograms after correction based on the mean defocus value. Scale bar is 100 Å.
Mentions: Using this approach, a map of PRD1 was obtained from uncorrected images with a resolution of 30 Å at the 3σ threshold and of 31 Å at the 0.5 threshold of the FSC (Fig. 5A, blue line).

Bottom Line: The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF.Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination.We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRD1 as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.

Show MeSH