Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy - PubMed (original) (raw)

Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

Anna-Clare Milazzo et al. J Struct Biol. 2011 Dec.

Abstract

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Conflicts of Interest

Prior to the research for this publication, A.-C.M. received consulting fees from Direct Electron for unrelated professional services.

Figures

Figure 1

Figure 1

Comparison of detection limit between cameras installed on the same microscope using identical instrument conditions and almost matching pixel size. Fourier transforms and radially averaged power spectra of 3K × 3K cropped images from: (A) DDD (1.38Å/pixel) and (B) CCD (1.37Å/pixel). High tension 200 KeV, electron dose ~20e−/Å2, exposure time 400ms. The modulation transfer function is shown in (C) for the CCD and DDD cameras calculated using the edge method.

Figure 2

Figure 2

Representative cropped 2000 × 2000 pixel micrographs of GroEL. (A) DDD image (1.38Å/pixel), (B) CCD image (1.37Å/pixel). Defocus value is approximately 2μm for both images. Scale bar is 40nm.

Figure 3

Figure 3

GroEL reconstructions from ~30K particle data sets comparing DDD in (A) and the CCD camera in (B). The final volumes with no post processing are shown in (a) and (e). Maps shown in (b) and (f) were amplitude corrected with an X-ray scattering curve out to 8Å and low pass filtered to 7.5Å. Volumes shown in (c) and (g) were amplitude corrected with an X-ray scattering curve out to 5Å and low pass filtered to 6Å. The Fourier Shell Correlation plots for (a) and (e) are shown in (d) and (h).

Figure 4

Figure 4

A high resolution DDD reconstruction from a 72,316 particle dataset is shown in (A), low pass filtered to 4.5Å. In (B), the Fourier Shell Correlation, derived from reconstructed even-odd particle data sets, gives the FSC0.5 as 6.1Å. For comparison, we show an asymmetric subunit from the DDD map in (C) and previously published asymmetric subunits from a 4Å map acquired using film in (D) (Ludtke et al., 2008) and a 5.4Å map from a CCD camera in (E) (Stagg et al., 2008).

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