Tilt series alignment and CTF correction

The tilt series were aligned with a marker-free method using the PROTOMO package (Winkler and Taylor, 2006). PROTOMO uses Radermacher’s weighted back-projection method (Radermacher, 2006).

The contrast transfer function (CTF) was determined for each tilt series image by averaging the power spectra from patches 256 × 256 pixels in size with a 50 pixel overlap along x and y axis (Huang et al., 2003). The average defocus value of the projection image was determined using the SPIDER command tf ed applied to the corresponding averaged power spectrum (Frank et al., 1996). Finally, a phase-flip CTF correction was calculated with the SPIDER command tf ct using the 0.8 cut off estimation. The CTF was measurable in individual images to 1.5 nm resolution.