Digitally collected cryo-electron micrographs for single particle reconstruction.
Several advantages and disadvantages have been cited for image collection with a slow-scan CCD camera. Here we explore its use for cryo-EM single particle reconstruction and present two practical examples. The icosahedral adenovirus (Ad) type 2 ( approximately 150 MDa) was reconstructed from 396 particle images. The Fourier shell correlation (FSC) 0.5 threshold and the Fourier shell phase residual (FSPR) 45 degrees criterion yielded 17 AA resolution for the ordered viral capsid. Visual comparison with the filtered Ad2 crystallographic hexon confirmed a resolution range of 15-17 A. The asymmetric DNA-PKcs protein (470 kDa) was reconstructed from 9,473 particle images, using a previously published reconstruction based on class-sum images as an orientational search model [Chiu et al. (1998) J. Mol. Biol. 284:1075-1081]. FSC and FSPR methods yielded 17 A 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 symmetry 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 significantly more DNA-PKcs images. Use of the CCD camera enables the microscopist to adjust the electron beam strength interactively and thereby maximize the image contrast for beam sensitive samples. On-line Fourier transformation also allows routine monitoring of drift and astigmatism during image collection, resulting in a high percentage of micrographs suitable for image processing. In conclusion, our results show that digital image collection with the YAG-scintillator slow-scan CCD camera is a viable approach for 3D reconstruction of both symmetric and asymmetric particles.
Stewart
P L
PL
Department of Molecular and Medical Pharmacology and Crump Institute for Biological Imaging, UCLA School of Medicine, Los Angeles, California 90095, USA. pstewart@mednet.ucla.edu
Cary
R B
RB
Peterson
S R
SR
Chiu
C Y
CY
D000260:Adenoviruses, Human / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000648:ultrastructure*; D012815:Signal Processing, Computer-Assisted
D004268:DNA-Binding Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases