Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy.
For three-dimensional (3D) structure determination of large macromolecular complexes, single-particle electron cryomicroscopy is considered the method of choice. Within this field, structure determination de novo, as opposed to refinement of known structures, still presents a major challenge, especially for macromolecules without point-group symmetry. This is primarily because of technical issues: one of these is poor image contrast, and another is the often low particle concentration and sample heterogeneity imposed by the practical limits of biochemical purification. In this work, we tested a state-of-the art 4 k x 4 k charge-coupled device (CCD) detector (TVIPS TemCam-F415) to see whether or not it can contribute to improving the image features that are especially important for structure determination de novo. The present study is therefore focused on a comparison of film and CCD detector in the acquisition of images in the low-to-medium ( approximately 10-25 A) resolution range using a 200 kV electron microscope equipped with field emission gun. For comparison, biological specimens and radiation-insensitive carbon layers were imaged under various conditions to test the image phase transmission, spatial signal-to-noise ratio, visual image quality and power-spectral signal decay for the complete image-processing chain. At all settings of the camera, the phase transmission and spectral signal-to-noise ratio were significantly better on CCD than on film in the low-to-medium resolution range. Thus, the number of particle images needed for initial structure determination is reduced and the overall quality of the initial computed 3D models is improved. However, at high resolution, film is still significantly better than the CCD camera: without binning of the CCD camera and at a magnification of 70 kx, film is better beyond 21 A resolution. With 4-fold binning of the CCD camera and at very high magnification (> 300 kx) film is still superior beyond 7 A resolution.
Sander
B
B
Research Group of 3D Electron Cryomicroscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Golas
M M
MM
Stark
H
H
D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000295:instrumentation*; D046911:Macromolecular Substances / Q000737:chemistry; D014027:Tobacco Mosaic Virus / Q000648:ultrastructure; D014771:Virion / Q000648:ultrastructure
D046911:Macromolecular Substances