Single-particle image processing
Cryo-EM images were digitised on a Zeiss SCAI photoscanner (ZI Imaging, Swindon, UK) with a pixel size of 7 μm. Cross-over repeats were selected by manually marking the cross-over positions using Ximdisp54,55 and were boxed out in SPIDER.56 Images were averaged to 0.35 nm/pixel at the specimen level and extracted into 1080 × 1080-pixel boxes for data processing. Defocus was determined from carbon adjacent to holes using Ctffind2,54,57 and image phases were corrected for the effects of the contrast transfer function using IMAGIC.58 Images were bandpass-filtered with a low-frequency cutoff between 70 and 20 nm (adjusted according to defocus) and a high-frequency cutoff of 1.16 nm and normalised. Images were aligned in SPIDER,56 with subsequent multivariate statistical analysis and classification of images in IMAGIC. An initial separation into the three structural types prior to classification was based on an analysis of the eigen images. Classes were all set to the same polarity. An iterative procedure of alignment, classification, and polarity reversal was performed until no classes of opposite polarity were obtained. Averaged power spectra of negative stain segments were obtained by multivariate statistical analysis and classification. The subunit repeat is easier to recognise in stained fibrils because of the higher contrast and as a consequence of single sided staining which avoids the overlap of upper and lower regions of the fibrils. This overlap obscures the subunit repeat pattern when the whole structure is seen in projection by cryo-EM because the repeats are out of register in the two halves of the fibrils.