Digitally collected cryo-electron micrographs for single particle reconstruction

Several advantages and disadvantages have been cited for image collection w ith a slow-scan CCD camera. Here we explore its use for cryo-EM single part icle reconstruction and present two practical examples. The icosahedral ade novirus (Ad) type 2 (similar to 150 MDa) was reconstructed from 396 particl e images. The Fourier shell correlation (FSC) 0.5 threshold and the Fourier shell phase residual (FSPR) 45 degrees criterion yielded 17 Angstrom A res olution for the ordered viral capsid. Visual comparison with the filtered A d2 crystallographic hexon confirmed a resolution range of 15-17 Angstrom. T he asymmetric DNA-PKcs protein (470 kDa) was reconstructed from 9,473 parti cle images, using a previously published reconstruction based on class-sum images as an orientational search model [Chiu et al. (1998) J. Mol. Biol. 2 84:1075-1081]. FSC and FSPR methods yielded 17 Angstrom resolution for the new DNA-PKcs reconstruction, indicating a small but noticeable improvement over that of the class-sum based reconstruction. Despite the lack of symmet ry for DNA-PKcs and its lower image contrast compared to Ad2 (0.8% vs. 2.5% ), the same resolution was obtained for both particles by averaging signifi cantly more DNA-PKcs images. Use of the CCD camera enables the microscopist to adjust the electron beam strength interactively and thereby maximize th e image contrast for beam sensitive samples. On-line Fourier transformation also allows routine monitoring of drift and astigmatism during image colle ction, resulting in a high percentage of micrographs suitable for image pro cessing. In conclusion, our results show that digital image collection with the YAG-scintillator slow-scan CCD camera is a viable approach for 3D reco nstruction of both symmetric and asymmetric particles. Microsc. Res. Tech. 49:224-232, 2000. (C) 2000 Wiley-Liss, Inc.