While the images in White et al, are nice, it has bothered me many times that the size of the shadow granules on the SP-D dodecamers in Figure 3A, and the CRD domains, look larger to me than those of B and C in the same figure. There is a bar marker of 100 nm in the top figure and while making micron bar markers is better than giving the magnification of the original micrograph as the only way to judge (measure) sizes of molecules, it seems to be fraught with error.  Here the CRD in part C of the figure are significantly different than the CRD in part A of the figure…so this needs to be explained.  Because part A of the figure is supposed to be the reference point (size), and no new bar markers are provided for parts B and C one is left with the need for some other way to determine an accurate length of the collagen-like domain and the neck portion and the Ntermini junctions of each of the molecules, and this appeared to me to be going back and measuring the grain size of the shadowing, and then remeasuring the CRD and collagen-like domain arm lengths.

n=80 CRD measured using a circle whose diameter covered the whole CRD as much as possible. In the gene-edited molecules of rSP-D, in particular the size of the CRD of the N-CRD molecules and the MINI-rSP-D was smaller. So what changes in the CRD….? or is this an editorial mistake. Red circle..diameter of mean size CRD in a rSP-D dodecamer — Gray circle, size of the CRD in the N-CRD (no collagen-like domains).

Also the means for the CRD are significantly different in these three groups, from one group to another. For example, N-CRD size against dodecamer CRD size. The CRD for the dodecamers is larger, this may be sample size error, which is just n=8 for the full size rSP-D CRD. The MINI-rSP-D is highly significantly different from the size of the rSP-D CRD. The t-value is -5.6584. The p-value is < .00001.  N=32 for the MINI-rSP-D, and the same 8 for the dodecamers that were analyzed against the diameters of the CRD on the N-CRD molecules (pink, n=80).

Because the size of the background grains is not significantly different in those three sets of images in White’s, Fig 3 (a,b,c) it seems unlikely it is a shadowing difference that causes the difference in CRD size among the three group: –  it might be a difference in the protein preparations in other respects. Images from other experiments would be required to verify.  The CRD of the N-CRD really do “look” different from the MINI-rSP-D, the latter looking to have the three individual CRD in a more compact configuration.

Shadowed grain size in a quick assessment, n=10 for each set of images, shows grains not seem to be significantly different in size moving from fig 3 a, to b, to c, at least with this random, but small, sample of 10 approximate grain diameters.