Figure 9. Atomic-scale model of the 32mer WCsAPF. (a-c) Rainbow-colored ribbon representation of the backbone. (a) View from the side. (b) Same as (a) except the outer and S3 strands have been clipped away to show the inner S1b-S2 helices surrounding the S1a 8-stranded β-barrel. (c) View down the central axis of the assembly. (d) Sphere representation of side chains colored by element for a central cross-section illustrating tight side chain packing. (e-g) Stick and ball side chains colored by element and backbone ribbon for the (e) top, (f) slightly lower, and (g) middle cross-sections.
DCsAPFs and their relationship to bAPFs
The second category of sAPFs, DCsAPFs, have a dark outer ring with cogs surrounding a light ring, which surrounds a dark circular center. Unlike the white-centered sAPFs, DCsAPFs with the same number of cogs can have different diameters (Fig. 10). Also, WCsAPFs and DCsAPFs of the same approximate diameters often have differing number of cogs and thus different symmetries; e.g., the putative 32mer WCsAPF has eight cogs and 8-fold radial symmetry, whereas the putative 32mer DCsAPF has four cogs and 4-fold radial symmetry. Comparison of sizes and shapes of DCsAPFs to those of bAPFs suggest that the number and distribution of DCsAPF cogs are like those of the beads of bAPFs. Most DCsAPF appear to have morphed from bAPFs composed of the two smallest sizes of beads; i.e., the ones we propose to be hexamers and octamers (outlined in purple and red in Figs. 2 and 10). If so, then the radial unit-cells should have six and eight monomers for the two principal groups of DCsAPFs.
The blue circles superimposed on the white rings of the DCsAPFs in Fig. 10 have the diameter predicted for a S3 β-barrel with the number of strands suggested by the number of monomers proposed for the associated bAPF; e.g., the bAPF composed of six small beads (hexamers) should have 36 monomers and the S3 β-barrel of the associated DCsAPF should have 36 β-strands. S/N values for these putative S3 β-barrels are 1.0 for the 18-32mers, and 2/3 or 3/4 for larger multiples of hexamers or octamers. Thus, these DCsAPFs are consistent with the hypothesis that an antiparallel S3 β-barrel is surrounded by a S1a-S1b-S2 β-barrel in which the S/N values change in a manner that maintains a gap distance between the walls of the barrels near 1.0 nm. It is difficult to predict based on these images whether there is a S1-S2 β-barrel inside the S3 barrel because the interior is completely dark. If the dark center is due to the presence of hexane, then hexane may shield the inner surface of the S3 β-barrel from water and any interior S1 and S2 segments may interact with the surface of a hexane layer.
An interesting exception to the general rule of relatively circular sAPFs are the putative oval-shaped 32mers proposed to have developed from diamond-shaped clusters of four octamers (7thimage of Fig. 10). Seven such images were identified in the portion of the micrograph of Fig. 1b that contains the WCsAPFs. These were the only oval sAPFs observed in this region of the EM and they all have similar sizes and shapes, and, like WCsAPFs, they have alternating dark and light layers with a white center. In this instance the sAPFs may have retained the general shape of its ancesteral bAPF rather than adopting β-barrel structures with circular cross-sections. The other possible exception are the DCsAPFs we propose to have 10-fold symmetry. Some of the isolated images appear to have 5-fold symmetry, which when radially averaged with 10-fold symmetry, would appear to have ten cogs. 5-fold symmetry could result if each radial unit-cell has 12 or 16 monomers instead of six or eight, which could occur if they developed from bAPFs composed of five dodecamers or hexadecamers.