 | Acta Crystallographica Section F Acta Crystallographica Section F STRUCTURAL BIOLOGY COMMUNICATIONS |
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![[Figure 1]](hv5346fig1mag.jpg)
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Figure 1
Schematic representation of three-dimensional single-particle reconstruction. The high purity of the sample is important, as in X-ray crystallography. Initially, the observation of the sample in negative staining is a useful step; as it clearly displays the sample, homogeneity can be checked. The particles are selected from the micrographs, centred and aligned. The classification and averaging images can improve the signal-to-noise ratio, and the class averages can be used to calculate an initial model at low resolution. This is calculated using the common-lines method or the random conical tilt series method (Dubochet et al., 1988  ). Then, in the cryo-EM step, the vitrified sample is imaged using a direct electron detector (a film is obtained from multiple images of the same field of the electron-microscope grid). Correcting for movement between the various images of the film is performed. The particles are then windowed and averaged. Determination of the defocus value allows correction of the contrast-transfer function (CTF). After alignment and classification of the data set, the orientation of each projected particle relative to the initial model is assigned. Refinement of the orientation values is performed iteratively until the three-dimensional structure of the macromolecule of interest converges (this figure was adapted from Boutin et al., 2016). |
![[Figure 1]](hv5346fig1.jpg)
| STRUCTURAL BIOLOGY COMMUNICATIONS |
ISSN: 2053-230X
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