C2H2-ZF domain divergence with respect to <i>D. melanogaster</i> reference.

<p>(A) Schematic of a C2H2-ZF protein—DNA interface under the 7-contact model [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005011#pgen.1005011.ref053" target="_blank">53</a>]. Amino acids within the depicted finger are numbered according to their relative position from the start of the alpha helix within the C2H2-ZF domain, with <i>a_-1</i> indicating the position before the start of the helix. Bases <i>b₁, b₂, b₃</i> and <i>b₄</i> are numbered sequentially from 5’ to 3’ of the primary DNA strand; the complementary bases are denoted by <i>b₁’, b₂’, b₃’</i> and <i>b₄’</i>. Contacts between amino acids and bases are shown in arrows, with four specificity-determining amino acids <i>a_-1, a₂, a₃</i> and <i>a₆</i> making these contacts. Solid arrows depict the four “canonical” contacts between ZF domains and DNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005011#pgen.1005011.ref052" target="_blank">52</a>], and dashed arrows depict three additional contacts that are used in our predictions of binding specificity [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005011#pgen.1005011.ref053" target="_blank">53</a>]. (B) Histogram showing the percent divergence per species by position within the C2H2-ZF domain’s alpha-helix (-1 to 6) for all canonically linked domains. The columns with blue labels in the <i>x</i>-axis correspond to positions that interact with DNA in the 7-contact model. (C) Percent of all (black), canonically linked (blue) and non-canonically linked (red) aligned domains in each non-reference fly species with a divergent residue (as compared to the <i>D. melanogaster</i> reference) in positions -1, 2, 3, and/or 6.</p

Conservation of predicted binding motifs for experimentally derived PWMs across species.

<p>(A) The list of analyzed experimentally determined C2H2-ZF binding specificity motifs (PWMs) within <i>D. melanogaster</i> along with a heat map representing the conservation of the corresponding protein construct across the fly species; note that each PWM was determined either for an entire protein or just a fragment of it. In the heat map, white depicts that a 1-to-1 ortholog for the corresponding C2H2-ZF protein in <i>D. melanogaster</i> was not present in that species; blue depicts that the DNA-contacting residues within the C2H2-ZF construct are conserved across all the flies; green depicts that the DNA-contacting residues within the C2H2-ZF construct did not diverge in that species, but one or more of these residues diverged in one or more orthologs in the other fly species; and orange depicts that the C2H2-ZF in the current species diverged from its 1-to-1 ortholog in <i>D. melanogaster</i> in at least one DNA-contacting residue within the protein construct. (B) For each species, ordered on the <i>x</i>-axis by its relative evolutionary distance from <i>D. melanogaster</i>, we plot for each PWM in panel A the fraction of promoters predicted to be bound in <i>D. melanogaster</i> whose orthologous regions within the species are also predicted to be bound. Blue points correspond to C2H2-ZFs conserved across all the flies, and orange points correspond to C2H2-ZFs that diverge in the current species. The medians of the conserved and diverged C2H2-ZFs for each species are computed and plotted as black points. Lines connecting these median points are drawn for visual effect only. For each species, conserved C2H2-ZF proteins tend to bind a higher proportion of promoter regions that are orthologous to those bound in <i>D. melanogaster</i> than do diverged C2H2-ZF proteins. (Part B Inset) Violin plots showing the per-species 0, 1-normalized ranks of percent orthologous promoter regions bound, such that the rank of the lowest percentage per species maps to 0, and the rank of the highest percentage maps to 1. The <i>p</i>-value comparing the normalized percentages between conserved and diverged C2H2-ZF orthologs is calculated using a Wilcoxon test. (C) Same as part B, where <i>y</i>-axis values correspond to the percent of high-confidence <i>D. melanogaster</i> binding sites conserved in each species for each PWM. For each species, conserved C2H2-ZF proteins tend to have a higher fraction of binding sites conserved from <i>D. melanogaster</i> than do diverged C2H2-ZF proteins.</p

Loss and recruitment of C2H2-ZF domains with respect to <i>D. melanogaster</i> reference.

<p>(A) Percent of <i>D. melanogaster</i> domains lost in each non-reference species for all domains (black) and separately for non-canonically linked (blue) and canonically linked (red) domains, with a phylogenetic tree relating the fly species to the left. (B) Percent of domains gained by each non-<i>melanogaster</i> species.</p

Example of a varying <i>D. melanogaster</i> C2H2-ZF domain.

<p>(A) Layout of the seven C2H2-ZF domains in <i>D. melanogaster</i> protein FBpp0072605. All domains are found in three canonically linked arrays of sizes 2, 2, and 3 respectively. Both domains in the middle array and domains 2 and 5 located at the end of the first array and start of the last array also exhibit divergent binding residues. (B) Closeup of the 4th domain in the protein, with phylogenetic tree and multiple alignment of the aligned domains from the other fly species. (C) Average (across positions b1–b4) Pearson correlation coefficients (PCCs) between non-reference and <i>D. melanogaster</i> SVM predicted specificities by species. The Spearman correlation, relating non-<i>melanogaster</i> predicted specificity change to phylogenetic distance from reference <i>D. melanogaster</i>, is also shown and implies that specificity changes increase gradually with distance from the reference. (D) Frequency plots of the PWMs generated by WebLogo [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005011#pgen.1005011.ref063" target="_blank">63</a>] representing unique binding specificities, predicted by the SVM method, ordered by phylogenetic distance from <i>D. melanogaster</i>, and labeled with the species whose domains had that corresponding binding specificity. Predicted positions with a PCC > 0.25 to one of either the ML or RF corresponding predictions are marked with a ×, and positions with a PCC > 0.25 to both the ML and RF corresponding predictions are marked with a *. (E) Distribution of Spearman correlations for each aligned domain (as in Part C) relating non-<i>melanogaster</i> predicted specificity change to phylogenetic distance from reference <i>D. melanogaster</i>. (F) Violin plots depicting the distributions of PCCs between predicted specificities for non-reference domains and their aligned domains in <i>D. melanogaster</i> orthologs.</p

Helical and non-helical organization of collagen.

<p>The non-helical, folded C-terminal end of the collagen molecule (top) extending from the triple-helical region (below). The electron density of neighboring collagen molecules can be seen along side the chain traced segment (red). The GPO₅ domain is indicated in white.</p

Greatly simplified organizational hierarchy of fibrillar collagen structure (from polypeptide to fibril)

<p>A. The collagen-forming polypeptide chains contain a large helix-forming domain with the repeat amino acid sequence Gly-X-Y, where X and Y are occupied by Pro or Hyp more frequently than other residues, but only account for approximately 1/6 of the total amino acid content (see for instance human sequence: ExPASy sequence data bank codes; P02452 and P08123). An arrow points to the figure element that shows that three polypeptides form the collagen monomer. The large triple-helix (super-helix) domain of approximately 300 nm in length is flanked by non-helical telopeptides (N and C, shown). The 6–8.6 nm dimension indicates the repeat of the triple-helix (36; 37). B. Collagen molecules are staggered approximately 67 nm from one another in the formation of microfibril aggregates. The microfibrils are D-periodic (D = 67 nm), and in each D-period, two monomers coil, or partially coil, around each other giving the appearance of another helix-like feature in the structural hierarchy (3). C. Cross-sectional view of the collagen molecular packing of a type I collagen fibril (11). Each circle represents one collagen molecule in cross-section (at the axial level of 0.44D). at the 0.44 D position. Next to B to C arrow, cross-section of an isolated microfibril. D) Archival image (Orgel laboratory) of the wide angle fiber diffraction pattern of type I collagen from rat tail tendon. The distinctly different but superimposed non-crystalline and crystalline diffraction patterns are indicated. Previous fiber diffraction studies of collagen's helical structure have concentrated on the non-crystalline part of the pattern, in this present study, we analyze crystalline diffraction data.</p

Patterson functions of collagen model structure factors 00L (meridional) series.

<p>A) Comparison of GPO (7/2 model) and GPO with collagen sequence threaded to check if amino acid sequence effects periodicities detected by the Patterson function. It does not appear so. B) As (A) except for GAA (10/3 model). C) Patterson functions of collagen types I and II are compared with those from the GAA and GPO coordinate models with the collagen sequence threaded onto them. The semi-transparent arrows mark: red, the maximum of the GAA (10/3) helix model pitch and repeat periods, the black arrows mark the collagen I and II respective positions for these periods. Note that the collagen experimental data show periods that are longer then the 7/2 and do seem to almost reach the 10/3 expected range. This could be interpreted to mean that both helical symmetries are found in native fibrillar collagen in addition to other possible conformations.</p

Patterson functions of the type I and II collagen 00L (meridional) series.

<p>A) Patterson function from 0.0–0.5D, the inverse (0.5–1.0) half of the Patterson function is not shown. The fractional distances between periodicities indicated in the functions has been multiplied by 67 nm (the length of the one dimensional unit cell – the D-period) for comparison with the helix symmetry periods. B) Enhanced view of the Patterson function range of interest for the helix symmetry periodicities. C) Table of key helix periodicities for comparison with A and B (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089519#pone-0089519-g004" target="_blank">Figure 4</a>).</p

Major structural features and ligand binding sites for collagen III.

<p>Major structural features and ligand binding sites for collagen III.</p

Arrangement of five overlapping collagen III α1 chains in the fibril D-period with overlap and gap zone indicated.

<p>Arrangement of five overlapping collagen III α1 chains in the fibril D-period with overlap and gap zone indicated.</p