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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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CTF fitted in the mean power spectrum of tilt series. The mean of rotationally averaged, background subtracted power spectra of all images of a tilt series were calculated (black lines). A CTF curve (dotted red line) was fitted using least square deviation methods. (A) Example from a tilt series of carbon film. The mean defocus value of the series is 3.5 μm. (B) Example from a cryo-tilt series of PRD1. The mean defocus value is 4 μm. The y axis in (B) is scaled up 20 times with respect to (A).
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fig3: CTF fitted in the mean power spectrum of tilt series. The mean of rotationally averaged, background subtracted power spectra of all images of a tilt series were calculated (black lines). A CTF curve (dotted red line) was fitted using least square deviation methods. (A) Example from a tilt series of carbon film. The mean defocus value of the series is 3.5 μm. (B) Example from a cryo-tilt series of PRD1. The mean defocus value is 4 μm. The y axis in (B) is scaled up 20 times with respect to (A).

Mentions: As shown in Fig. 2A, for tilt series which reflect the conditions of real data collection, the curve which describes the mean of the CTFs in a tilt series approximately overlaps at low frequencies with the CTF of a single image at the mean defocus value. This suggests that the standard method for defocus determination in cryo-EM: fitting the curve described by Eq. (1.0) to the mean of the rotationally averaged power spectra of the tiles, could be applied to the tomographic series if sufficient signal was present, and if only the low frequency region of the curve was considered. We tested this on a number of tilt series of a carbon film. The mean defocus value for a tilt series was determined by fitting a standard CTF curve to the mean of the power spectra using a least square curve fitting algorithm (Fernando, personal communication). One example is shown in Fig. 3A, where a CTF curve is fitted to the experimental data obtained by calculating the mean of the power spectra of images in a tilt series and subtracting the background. The defocus value is 3.4 μm. The mean defocus value of individual images from the same tilt series measured using the EMAN program CTFIT (Ludtke et al., 1999) is 3.3 μm. Considering 10 tilt series the error in the defocus estimate was 0.3 ± 0.2 μm.


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)

CTF fitted in the mean power spectrum of tilt series. The mean of rotationally averaged, background subtracted power spectra of all images of a tilt series were calculated (black lines). A CTF curve (dotted red line) was fitted using least square deviation methods. (A) Example from a tilt series of carbon film. The mean defocus value of the series is 3.5 μm. (B) Example from a cryo-tilt series of PRD1. The mean defocus value is 4 μm. The y axis in (B) is scaled up 20 times with respect to (A).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: CTF fitted in the mean power spectrum of tilt series. The mean of rotationally averaged, background subtracted power spectra of all images of a tilt series were calculated (black lines). A CTF curve (dotted red line) was fitted using least square deviation methods. (A) Example from a tilt series of carbon film. The mean defocus value of the series is 3.5 μm. (B) Example from a cryo-tilt series of PRD1. The mean defocus value is 4 μm. The y axis in (B) is scaled up 20 times with respect to (A).
Mentions: As shown in Fig. 2A, for tilt series which reflect the conditions of real data collection, the curve which describes the mean of the CTFs in a tilt series approximately overlaps at low frequencies with the CTF of a single image at the mean defocus value. This suggests that the standard method for defocus determination in cryo-EM: fitting the curve described by Eq. (1.0) to the mean of the rotationally averaged power spectra of the tiles, could be applied to the tomographic series if sufficient signal was present, and if only the low frequency region of the curve was considered. We tested this on a number of tilt series of a carbon film. The mean defocus value for a tilt series was determined by fitting a standard CTF curve to the mean of the power spectra using a least square curve fitting algorithm (Fernando, personal communication). One example is shown in Fig. 3A, where a CTF curve is fitted to the experimental data obtained by calculating the mean of the power spectra of images in a tilt series and subtracting the background. The defocus value is 3.4 μm. The mean defocus value of individual images from the same tilt series measured using the EMAN program CTFIT (Ludtke et al., 1999) is 3.3 μm. Considering 10 tilt series the error in the defocus estimate was 0.3 ± 0.2 μm.

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