This might end up providing clues as to where the center of the dodecamer divides the arms on the acute angles. It relies on a common point counting method in morphometry and the readings on grey scale measurements using imageJ. It will take some handwork, no question, if you know an more automated way. please advise.

Another option worked on today is to cut the molecules and moving the parts into a straight line, then cutting a small band through the center where the variations in grey scale are found and using ImageJ to plot the grey scale values, setting up a plot in excel and using that under neath the visual.  I used photoshop at half transparency to put the right arms and left arms over each other and increasing the contrast of the two, then flattening the image and exporting a tiff to open in ImageJ.  The best rectangle through the dodecamer then is a blend of the two arms on the 45 degree angles (purposefully to see if there is a size and density pattern to the center part of the dodecamer that might shed light on how many of the amino acids in the collagen-like portion are involved there.

The same dodecamer as seen above, is the top image, then the “straightened image (i used about 25 cuts per arm) is shown for the two by two arms of the dodecamer below, and the blended half transparent flattened image is the the thin band just above the grey scale density plot.  Clearly there is a central area….matching that in the top image, it will take some additional images to find out whether the peaks along the arms are consistent or just random. One thing for sure, this plot does NOT show the definite bands that the image from Arroyo et al did.  I see that they used one particular image and this one i will test next.

Adding to this is a sample from which all four arms are leveled out using corel draw and/or photoshop and aligned to a common length (CRD to N terminal) relative to each other, with two arms mirrored so that all CRD are pointing the same direction, each image overlay was taken to 80% or so transparency, the image flattened and a exported as a tiff and then opened in ImageJ for a grey-scale intensity plot. The two plots (upper one that is two arms blended in a dodecamer, and lower one, 4 arms blended and mirrorred) still give the impression that the peaks are related to the density of the particles shadowing the molecule. THe clearest differences are found in the size of the most central part of the N terminal and the outer globs of the CRD. It is possible that NO pattern can be determined from this approach.

The best test for the background is to do some plots on areas that are adjacent to each of the molecules.

He is one such LUT plot for a background from the same group as is dodecamer above and the magnifications are relative to each other. The background has a 25 or 26 peak to peak distance in 100nm where the molecule that is shadowed has about 16, or about 5 in the images by Arroyo et al.  Neither is probably correct.  The peak height in the background is not the same as the dodecamers either, though images were not manipulated together, so that is another control to do.

It is pretty obvious that the center of SP-D (at least as is represented in dozens and dozens of TEMs, and AFMs ) is longer in the direction of the 45 degree angle than the amino acid sequence would suggest and longer too than most diagrams present. There is a central dense region (which is found also on the tracing) but an extended region where two arms of the dodecamer are closely aligned with the combined length of something around 30nm, or a little less than a third of the entire length of the dodecamer.  The separation of the two arms on either end into the @45 degree angle  leaves the remainder of the arms as the collagen-like domain, and the neck and CRD on the order of 10-13nm the latter folded back upon the neck section to some degree. Tracing from Figure 2. Arroyo et al. JMB. 430, 1495-1509, 2018. Their measurement for the total width of a surfactant dodecamer seemed to be considerably larger than measured by others. So a compromise in estimated dimension has to be taken because of the differences in measurement accuracy and how much spreading of the molecules has occurred…  Relative measurements then, of 100 nm might be useful in determining the relative lengths of the segments of the dodecamer, but not the absolute lengths.

Normalizing the dodecamers to 100nm cutting centrally through at least two (probably best making it two polar) CRD is useful. A summary of the center (the highest, brightest (by TEM and AFM) areas of the SP-D dodecamer might be about

I would like to find a way to trace density of a curved line…. straight lines are no problem.  I think it is time to go back and measure these all one more time but setting density and width standards to get relative dimensions which at some point could be used for generalities describing proportions and regions involved in SP-D. s

After figuring out that the images from one particular publication are between 15 and 20% larger than the micron marker accompanying the figure indicates, I went back to re-determine how much dodecamer arm length that the mini-SP-D lost when exons 3 and 4 were deleted from the collagen domain.  Again measuring the full SP-D in the micrograph and then using other images of SP-D and their respective bar markers I think that after several sets of measurements the miniSP-D has been reduced to about 35% of its original arm length. I am measuring the center portion (which surely contains more amino acids than just the N terminal) where it bifurcates.  Different sets of measurements sort of come in around 25 or 6 nm. The CRD are pretty close, and measurements indicate that the top left figure (which is the one which has the out of sorts micron marker (indicated by the different measurements for the diameter of the  underlying SP-D dodecamer… the red circle being what was given as 100nm (clearly too short) and the green circle indicating a diameter that would intersect the CRD in 3 different locations (typically I have used intersection of 2, but this one has three by chance) and is a much more accurate indicator of a 100nm.  Using the later, the center is N term plus whatever of the collagen portion is associatiated) is about 26 hm..  in the full length AND the mini-SP-D  so this suggests that the measurements are more in line, and also that the central X of the dodecamer hasn’t changed much with the deletion of the C3 and C4 regions.   Bars used to measure the arm length are the thin black lines in the lower right.

I am not putting these numbers out as anything but approximate, but they are similar to earlier posts, and a general indication of size.

One thing that is kind of interesting to me is that the collagen-like domain is often referred to as “disorganized” and often referred to as “braided” or like the helix of collagen.  There really should not be much discussion on whether it is organized or not… haha…  just looking at the pictures.