## 2 Results and Discussion
### Interpretations from AFM and XRD Studies  
Both AFM and XRD data show D-periodic binding of the probe after 1-2 h of label incubation. AFM shows a repeating pattern (Figures 2 and 3) with what appears to be a single labeling site per D-period. X-ray data appear to support this, as the difference Fourier indicates a major binding site at 0.42D and two to three minor binding sites at around 0.32, 0.85, and possibly at 0.99D (see Figure 5 and Table 4). Figure 4: Low angle X-ray diffraction of native and antibody-labeled type I collagen tendon samples, meridional orders 1–12. Scaled to same global maximum for relative intensity comparison. (**A**) Native rat tail tendon; (**B**) Anti-collagen type I antibody-labeled rat tail tendon. Figure 5: \(Cont\). A derivative labeling model built on this assumption (Figure 5A), produces a Patterson that significantly resembles the difference [PERSON] calculated from the experimental data Figure 5B). Both show the site-to-site spacing between antibody probes within a D-period. A large non-zero peak of 0.43D and a lesser peak at 0.10D. This is the distance between 0.99/0.1 and 0.42 and 0.85D and the 0.32 and 0.42D antibody labeling sites, respectively (as indicated from the difference Fourier). Both the Fourier and Patterson functions of the XRD data indicate that the major labeling site is at 0.42D, which is specifically at the start of the C-telopeptide. This would be the most exposed/elevated (i.e., presented on the outermost part of the fibril into the milieu of the matrix) part of the D-periodic fibril surface. This could be part of the reason why this is easily and prominently detected via AFM. This observation is in line with the site of recognition (epitope) for the antibody reported by manufacturers of anti-collagen I antibodies (COL1A1 aa. 1200-1300; see abcam Cat No. ab138492). It is interesting that this is the site of collagen polymerization and the site of GPVI and ENDO180 and OSCAR \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Fractional Peak Position**} & \multirow{2}{*}{**Fibril Accessibility**} & **Candidate Amino Acid** & **Functional Binding Region** \\  & & **Sequences (Monomer & **of Collagen D-Period** \\  & & **(M) and Sequence)** & **of Collagen D-Period** \\ \hline \multirow{2}{*}{0.31–0.33} & Just prior to C-term & M4 GPP(2) & Near (N-terminal) \\  & telopeptide. M4, M5 & M5 GPPGPR & fibronectin binding site (M4) \\ \hline \multirow{3}{*}{0.42–0.44} & Interdomain, & \multirow{3}{*}{M4 GVVGLPCQRGER} & Middle of the fibronectin \\  & helical/non-helical of M5 & & M5 GPO(orP)(5) & binding/MMP interaction \\ \cline{1-1}  & near C-term telopeptide. & & SGY/YYR & domain (M4) \\ \hline \multirow{3}{*}{0.84–085} & Highly accessible area in & \multirow{3}{*}{M4 GPP(GAP)3 GPVGPA} & Directly over DDR/VWF on \\  & gap region, just before & & \\ (\(n\)-terminal) decoron & & & M3 and adjacent to decoron \\ \cline{1-1}  & d-band binding site & & binding on M4 \\ \hline \multirow{3}{*}{0.99–0.1} & Lowest trough of & \multirow{3}{*}{M4} & \multirow{3}{*}{Near N-terminal telopeptide,} \\  & the up/down formation & & \\ \cline{1-1}  & of the collagen fibril & GPRCDK/GSRGAAGPP & possible decorin binding \\ \cline{1-1}  & surface, near N-term & M5 GPRCDK/GETGEQ & region \\ \cline{1-1}  & telopeptide & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Antibody binding positions. Figure 5: **(A) Difference Fourier/electron density map, showing the labeling positions in the antibody treated sample; (B) Difference Patterson, showing the peak occurring distance functions between antibody labeling positions. There is a clear correlation between the observed and calculated Patterson, which was based entirely on the observed peaks in the difference Fourier. These were the following: 0/0.99D, 0.31–0.33D, 0.42D–0.44D, and 0.85D. **  
MHC1 class collagen binding molecules. Presumably this is due to the repeating modified imino peptide sequence, GPO5, at this location.