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Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6.

Grant T, Grigorieff N - Elife (2015)

Bottom Line: Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution.At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image.This 'high-exposure' technique should benefit cryo-EM work on all types of samples, especially those of relatively low-molecular mass.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image. Using a 2.6 Å resolution single particle cryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of 100 electrons/Å(2), we obtained accurate measurements of optimal exposure values over a wide range of resolutions. At low and intermediate resolutions, our measured values are considerably higher than obtained previously for crystalline specimens, indicating that both images and movies should be collected with higher exposures than are generally used. We demonstrate a method of using our optimal exposure values to filter movie frames, yielding images with improved contrast that lead to higher resolution reconstructions. This 'high-exposure' technique should benefit cryo-EM work on all types of samples, especially those of relatively low-molecular mass.

No MeSH data available.


Related in: MedlinePlus

Surface rendering of an isolated small helix from different 3-frame reconstructions shown with the docked model.Each reconstruction is shown with its exposure range and resolution as calculated from the FSC using the 0.143 cut-off.DOI:http://dx.doi.org/10.7554/eLife.06980.008
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fig6: Surface rendering of an isolated small helix from different 3-frame reconstructions shown with the docked model.Each reconstruction is shown with its exposure range and resolution as calculated from the FSC using the 0.143 cut-off.DOI:http://dx.doi.org/10.7554/eLife.06980.008

Mentions: The first 27 frames of the movie were excluded from the analysis so as to prevent beam-induced movement from interfering with the results. Our results are therefore based upon data, which have effectively been pre-exposed by ∼20 e−/Å2. If radiation damage indeed occurs as a single-exponential process, pre-exposure should have no effect on the result presented here. We cannot exclude the possibility that the damage may occur in two (or more) phases and that we are only observing the final phase, but there is no evidence for a multi-phased process, and previous studies on crystalline specimens also demonstrate a single-exponential decay. Moreover, for lower resolutions at which beam-induced movement is expected to have less of an effect, a plot of ln(SNR) vs accumulated exposure across all frames shows a single-exponential process (see Figure 4). We also note that a reconstruction calculated using only frames 25–27 (∼19 e−/Å2–∼21 e−/Å2) still yields a 3.35 Å resolution reconstruction with clear side chain density (see Figure 6), suggesting that the decay we have measured is relevant to the kinds of structural information we are interested in. As expected, the density of side chains fades with increasing exposure and dose deposited on the sample. The density of carboxyl groups (e.g., Asp29, Figure 6) fades most rapidly and is already partially gone after an exposure of about 3 e−/Å2, while those of aromatic groups (e.g., Tyr24, Figure 6) persists even after an exposure of about 35–40 e−/Å2. This general pattern agrees with that observed before in a number of cryo-EM (Allegretti et al., 2014; Bartesaghi et al., 2014; Fromm et al., 2015) and X-ray crystallography studies (Fioravanti et al., 2007). We note also that the density of the α-helix backbone remains visible even after an exposure of ∼50 e−/Å2. This high exposure exceeds the optimal exposure of about 30 e−/Å2 at 8 Å resolution where α-helical features become clearly visible. Such high tolerance of secondary structural features to radiation damage may help in the alignment of single-particle images recorded using high exposures (see below).10.7554/eLife.06980.008Figure 6.Surface rendering of an isolated small helix from different 3-frame reconstructions shown with the docked model.


Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6.

Grant T, Grigorieff N - Elife (2015)

Surface rendering of an isolated small helix from different 3-frame reconstructions shown with the docked model.Each reconstruction is shown with its exposure range and resolution as calculated from the FSC using the 0.143 cut-off.DOI:http://dx.doi.org/10.7554/eLife.06980.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471936&req=5

fig6: Surface rendering of an isolated small helix from different 3-frame reconstructions shown with the docked model.Each reconstruction is shown with its exposure range and resolution as calculated from the FSC using the 0.143 cut-off.DOI:http://dx.doi.org/10.7554/eLife.06980.008
Mentions: The first 27 frames of the movie were excluded from the analysis so as to prevent beam-induced movement from interfering with the results. Our results are therefore based upon data, which have effectively been pre-exposed by ∼20 e−/Å2. If radiation damage indeed occurs as a single-exponential process, pre-exposure should have no effect on the result presented here. We cannot exclude the possibility that the damage may occur in two (or more) phases and that we are only observing the final phase, but there is no evidence for a multi-phased process, and previous studies on crystalline specimens also demonstrate a single-exponential decay. Moreover, for lower resolutions at which beam-induced movement is expected to have less of an effect, a plot of ln(SNR) vs accumulated exposure across all frames shows a single-exponential process (see Figure 4). We also note that a reconstruction calculated using only frames 25–27 (∼19 e−/Å2–∼21 e−/Å2) still yields a 3.35 Å resolution reconstruction with clear side chain density (see Figure 6), suggesting that the decay we have measured is relevant to the kinds of structural information we are interested in. As expected, the density of side chains fades with increasing exposure and dose deposited on the sample. The density of carboxyl groups (e.g., Asp29, Figure 6) fades most rapidly and is already partially gone after an exposure of about 3 e−/Å2, while those of aromatic groups (e.g., Tyr24, Figure 6) persists even after an exposure of about 35–40 e−/Å2. This general pattern agrees with that observed before in a number of cryo-EM (Allegretti et al., 2014; Bartesaghi et al., 2014; Fromm et al., 2015) and X-ray crystallography studies (Fioravanti et al., 2007). We note also that the density of the α-helix backbone remains visible even after an exposure of ∼50 e−/Å2. This high exposure exceeds the optimal exposure of about 30 e−/Å2 at 8 Å resolution where α-helical features become clearly visible. Such high tolerance of secondary structural features to radiation damage may help in the alignment of single-particle images recorded using high exposures (see below).10.7554/eLife.06980.008Figure 6.Surface rendering of an isolated small helix from different 3-frame reconstructions shown with the docked model.

Bottom Line: Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution.At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image.This 'high-exposure' technique should benefit cryo-EM work on all types of samples, especially those of relatively low-molecular mass.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image. Using a 2.6 Å resolution single particle cryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of 100 electrons/Å(2), we obtained accurate measurements of optimal exposure values over a wide range of resolutions. At low and intermediate resolutions, our measured values are considerably higher than obtained previously for crystalline specimens, indicating that both images and movies should be collected with higher exposures than are generally used. We demonstrate a method of using our optimal exposure values to filter movie frames, yielding images with improved contrast that lead to higher resolution reconstructions. This 'high-exposure' technique should benefit cryo-EM work on all types of samples, especially those of relatively low-molecular mass.

No MeSH data available.


Related in: MedlinePlus