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spider - align pairwise
This protocol provides the wrapper for the another variation  of reference-free alignment algorithm in SPIDER. It is based on OR SH and AP SH programs and performs both translational and rotational alignment.
Conceptually, this alignment first aligns pairs of images and averages them (fig. 1b). Then, it aligns pairs of averages of those pairs and averages them, and so forth. This type of alignment appears to be less random than does AP SR, which chooses seed images as alignment references.
User has to provide several input parameters (fig. 2):
Input particles should have positive contrast, i.e. objects should be bright on the dark background.
Similarly to AP SR protocol, you have to decide the limits of integration for the rotational search (in terms of inside and outside rings). The outer radius has to correspond to the particle size. To avoid oversampling (and thus the relatively large error of interpolation) an inner radius of 5 px was found to be sufficient for all practical purposes.
The average will be centered before a a final alignment. One centering strategy uses the SPIDER command CG PH. This command sometimes fails, so another strategy is to rotate the particle by 180 degrees and align it to itself. Sometimes, both of these strategies are worse than doing nothing.
For the translational alignment you have to provide the shift range and step in pixels.
When the protocol is finished you may click on the Analyze Results button. The produced output are the final total average and a stack of aligned particles (Fig. 3).
[[]] Sergio Marco, Mónica Chagoyen, Luis Gerardo de la Fraga, Jose María Carazo, Jose L Carrascosa. A variant to the “random approximation” of the reference-free alignment algorithm. Ultramicroscopy (1996), Volume 66, Issues 1–2, Pages 5–10. DOI