It is not sufficient just to count the number of peaks in an AFM image of surfactant protein D. That sounds like a rediculous comment but the signal processing programs do just that.  Provide a number of peaks. This just doesn’t help when there are subtle peaks, very prominent peaks, differences in peak width and height, some relative to the height of other peaks and gross variations in the way the trimer (three loosly or tightly bound monomers, depending upon which domain you are looking at and the CRD even with areas which bend over the neck domain in the images which changes the plot lines dramatically.

Similarly, the N termini junction has been  noted as having two different ways for the trimers to assemble into multimers and this too is shown in plots (sometimes two (or three) peaks at the top of the N term grayscale peaks), sometimes just 1 peak.  This likely will sort out in two ways 1) whether the line plotting grayscale goes through a side of the N term junction or through the “center” the latter likely not always “up” in the image.

Sorting the peaks into at least four different categories is necessary when counting up the number of peaks.  In this study, the N termini peak is counted from its valley most distant from the CRD through the entire N term-peak, UNLESS there are TWO distinct peaks (counted by the signal processing algorithms, in which case each half of the whole N term is counted separately.

That way an entire N term length is counted for each trimer, actually with a grayscale plot which always is recorded from N to CRD (regardless of the direction that the protein lies in the image).  The actual line CRD to CRD but the width and peak heights are recorded in the database in order that all calculations can be calculated on trimers, and all calculations begin with N and end with the CRD.