Figure 5. The procedure for defining the defocus of a tilted image.

(A) The whole micrograph is divided into 8 × 8 mosaic tiles. (B) Each sub-image was used to measure the defocus after Fourier transfer. (C) Though a modified power spectrum (PS) of the sub-image, (D) the curve was compared with the theoretical contrast transfer function (CTF) curve via the calculation of the cross-correlation within a defined frequency range. (E) By screening the defocus within a searching range, the defocus which maximized the cross-correlation (CC) between the modified experimental curve and theoretical curve was used as the measured defocus for this sub-image. (F) By repeating the above procedure on each of the other sub-images, we obtained the defocus distribution against their whole micrograph. (G) The distribution can be fitted with a linear gradient distribution plane. (H) By subtracting this plane from the original 2D distribution, we could obtain a distribution of the residuals for defining the “bad” defocus measurements within the sub-images. The bad measured defocus was defined as any residual above twice the standard deviation (such as the two defocuses marked in red crosses). After the bad defocus tiles are removed, the defocus distribution was then re-fitted a linear distribution plane, and the defocus at the center was used as the defocus of the tilted micrograph.