We first processed the set of 65 protein sequences (21 30S subunits, 33 50S subunits, and 11 translation accessory factors) in Aligator to obtain up to 1000 top-ranked strategies (i.e., sets of possible segments divided at valid ligation junctions) for each protein. Using our original default program settings for valid ligation junctions (restricted to junctions between an acceptable thioester aa [A, C, F, G, H, I, K, L, M, R, S, T, V, W, or Y] and thiol aa [C or A]) and a reasonable segment length limit (60 aa, less generous than in our previous report), we were unable to find valid strategies for 5 of the 65 proteins, as the gap between possible ligation junctions in these sequences was too large. Thus, we modified the program to accept additional thiol sites, which required substantial improvements to Aligator's processing capability to handle the increased number of possible segment combinations (see Supplementary Methods). For this analysis, we chose to include V as an acceptable 3 rd thiol site, as penicillamine (Pen, or β-thio-5 valine) is commercially available as an Fmoc-SPPS building block (in L or D) and can be desulfurized simultaneously with Ala. Pen ligations typically suffer from extremely hampered ligation kinetics and thus are penalized more so than poor thioesters in both Aligator and BracketMaker scoring algorithms (poor thioesters paired with Pen receive combined penalties). With an expanded list of possible ligation sites (allowing C, A, and V thiols) and a 60-aa 10 segment limit, we obtained valid strategies for all 65 ribosomal proteins ranging from 1-22 segments. Using the Aligator Import tool in BracketMaker, we applied Auto-Bracket to assemble each set into the most efficient calculated bracket with settings reflecting the most widely used CPS techniques. Assuming peptides are initially made as hydrazides, we chose to use Acm as the internal Cys protecting group and Tfa-Thz as the N-terminal Cys protecting group, allowing one-15 pot deprotection of N-terminal Cys. From the resulting list of up to 1000 brackets per protein, we identified the top overall bracket (smallest Max and Avg path lengths) for each (Figure ). Initial Assessment of Ribosome Brackets Due to differences in the way Aligator and BracketMaker rank strategies (e.g., Aligator does not account for ligation order, and BracketMaker does not account for segment length or solubility), the top Aligator strategy differs from the best bracket in approximately half of cases within this ribosomal set, especially in larger strategies. In the protein L5, for example, the winning Aligator strategy uses four segments of ideal length (30-50 aa each, 179 aa total) joined at C, A, and V ligation sites with a Max Path of 6, with the V ligation contributing a +2 path penalty. In contrast, the winning L5 bracket in BracketMaker uses entirely A ligation sites for a significantly improved Max Path of 4, but the segments (three >50 aa and one 12 aa) score poorly in Aligator because of their lengths.