Jose Miguel de la Rosa Trevin edited this page May 21, 2016 · 2 revisions

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spider - align pairwise

This protocol provides the wrapper for the another variation [3] 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.

00.SpiderProtAlignPairwise diagram
Figure 1. Block diagram of the algorithms for initial average calculation: (a) - standard reference-free algorithm, (b) - pairwise algorithm used in this protocol. In the two cases i are the input images and F is the final average image

Input parameters

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.

Figure 2. GUI input form of the spider - align pairwise protocol

Analyzing results

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).

02.SpiderProtAlignAPSR Results
Figure 3. Displaying alignment results


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