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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

(A) Example of an aligned movie sum of the rotavirus double-layered particle (DLP) sample imaged with a total exposure of 100 e−/Å2.(B) Particle sum created using all frames and (C) the first 3 frames of the movie, demonstrating the kind of data used in the analysis. In all cases, the scale bar represents 500 Å.DOI:http://dx.doi.org/10.7554/eLife.06980.003
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fig1: (A) Example of an aligned movie sum of the rotavirus double-layered particle (DLP) sample imaged with a total exposure of 100 e−/Å2.(B) Particle sum created using all frames and (C) the first 3 frames of the movie, demonstrating the kind of data used in the analysis. In all cases, the scale bar represents 500 Å.DOI:http://dx.doi.org/10.7554/eLife.06980.003

Mentions: Examples of an aligned movie and particle images with different effective exposures are shown in Figure 1. Processing exposure-filtered images (see below) of ∼4000 rotavirus particles and exploiting both their icosahedral and the 13-fold non-icosahedral symmetry of the outer layer led to a reconstruction of the VP6 trimer with clear density for side chains as well as the central Zn and Cl ions and a number of water molecules (Figure 2, Video 1 and 2). At the estimated resolution of 2.6 Å (Figure 3A), atomic model building is possible (Grigorieff and Harrison, 2011) and an atomic model obtained by refinement of a previously published structure (Mathieu et al., 2001) agrees well with the starting model as indicated by an RMSD of 0.4 Å. Despite good overall agreement, there are a number of significant differences, localized mainly to areas on the periphery of the molecule, which may indicate effects of crystal packing and/or the presence of additional protein interactions in the full capsid as opposed to the isolated VP6 subunit.10.7554/eLife.06980.003Figure 1.(A) Example of an aligned movie sum of the rotavirus double-layered particle (DLP) sample imaged with a total exposure of 100 e−/Å2.


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

Grant T, Grigorieff N - Elife (2015)

(A) Example of an aligned movie sum of the rotavirus double-layered particle (DLP) sample imaged with a total exposure of 100 e−/Å2.(B) Particle sum created using all frames and (C) the first 3 frames of the movie, demonstrating the kind of data used in the analysis. In all cases, the scale bar represents 500 Å.DOI:http://dx.doi.org/10.7554/eLife.06980.003
© Copyright Policy
Related In: Results  -  Collection

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

fig1: (A) Example of an aligned movie sum of the rotavirus double-layered particle (DLP) sample imaged with a total exposure of 100 e−/Å2.(B) Particle sum created using all frames and (C) the first 3 frames of the movie, demonstrating the kind of data used in the analysis. In all cases, the scale bar represents 500 Å.DOI:http://dx.doi.org/10.7554/eLife.06980.003
Mentions: Examples of an aligned movie and particle images with different effective exposures are shown in Figure 1. Processing exposure-filtered images (see below) of ∼4000 rotavirus particles and exploiting both their icosahedral and the 13-fold non-icosahedral symmetry of the outer layer led to a reconstruction of the VP6 trimer with clear density for side chains as well as the central Zn and Cl ions and a number of water molecules (Figure 2, Video 1 and 2). At the estimated resolution of 2.6 Å (Figure 3A), atomic model building is possible (Grigorieff and Harrison, 2011) and an atomic model obtained by refinement of a previously published structure (Mathieu et al., 2001) agrees well with the starting model as indicated by an RMSD of 0.4 Å. Despite good overall agreement, there are a number of significant differences, localized mainly to areas on the periphery of the molecule, which may indicate effects of crystal packing and/or the presence of additional protein interactions in the full capsid as opposed to the isolated VP6 subunit.10.7554/eLife.06980.003Figure 1.(A) Example of an aligned movie sum of the rotavirus double-layered particle (DLP) sample imaged with a total exposure of 100 e−/Å2.

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