Achievable resolution from images of biological specimens acquired from a 4k×4k CCD camera in a 300-kV electron cryomicroscope

Abstract

Bacteriorhodopsin and ε 15 bacteriophage were used as biological test specimens to evaluate the potential structural resolution with images captured from a 4k × 4k charge-coupled device (CCD) camera in a 300-kV electron cryomicroscope. The phase residuals computed from the bacteriorhodopsin CCD images taken at 84,000 × effective magnification averaged 15.7° out to 5.8-Å resolution relative to Henderson's published values. Using a single-particle reconstruction technique, we obtained an 8.2-Å icosahedral structure of ε 15 bacteriophage with the CCD images collected at an effective magnification of 56,000 ×. These results demonstrate that it is feasible to retrieve biological structures to a resolution close to 2/3 of the Nyquist frequency from the CCD images recorded in a 300-kV electron cryomicroscope at a moderately high but practically acceptable microscope magnification.

Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency

“…We have previously used ε15 bacteriophage to assess the performance of a variety of CCD detectors (Bammes et al, 2011; Chen et al, 2008; Booth et al, 2006). We acquired 75 frames of frozen, hydrated ε15 bacteriophage at 20,000× detector magnification, yielding sampling of 3.00 Å/pixel.…”

“…Since the performance of an electron detector depends on its acquisition of signal and noise, the spectral SNR under typical experimental conditions is the most pragmatic method for performance evaluation. Such practical characterizations have been vital to the development and acceptance of CCD cameras for cryo-EM imaging (Bammes et al, 2011; Chen et al, 2008; Booth et al, 2006; Sander et al, 2005; Booth et al, 2004; Zhang et al, 2003; Downing and Hendrickson, 1999). …”

“…Although CCDs provide increased low-frequency contrast (Booth et al, 2006; Sander et al, 2005), their detective quantum efficiency (DQE) at medium and high spatial frequencies is significantly lower than for photographic film, resulting in lower resolution images (McMullan et al, 2009). In the best practical cases, a typical CCD camera with 15 µm pixel size can record usable signal up to ~2/3 Nyquist frequency (Zhang et al, 2010; Chen et al, 2008). Unfortunately, new CCD cameras with smaller pixel sizes have not resulted in improved resolution, suggesting that current CCD technology for electron detection may have reached its limit (Bammes et al, 2011).…”

“…Our laboratory has previously evaluated the practical utility of digital imaging devices for low dose cryo-EM, including a 16 megapixel CCD camera (Chen et al, 2008; Booth et al, 2006) and a 111 megapixel CCD camera (Bammes et al, 2011). Here, we present a similar practical performance evaluation of a CMOS-based direct electron detector.…”

CTER—Rapid estimation of CTF parameters with error assessment

“…The more recent trend, as proposed by designers of Appion, is to integrate data collection automation with single particle structure determination packages so that a full project could in theory be completed with minimal user intervention [7]. This progress is greatly aided and accelerated by the development of a new generation of digital cameras whose large field, excellent detective quantum efficiency (DQE), and speed of readout all but eliminates the need for film as a data collection medium, thus further facilitating automation [8,9]. Of particular importance is the emergence of affordable direct detection device (DED) camera systems [10–13] that makes it possible to correct for beam-induced specimen motion [14–16], particularly when used in electron counting mode, and to carry out atomic resolution cryo-EM on a routine basis [17,18].…”