"Hard" Proteins (Max Path 5-6) As proteins increase in size and number of segments, challenges compound. The "Hard" category (12 ribosomal proteins) spans the largest range in terms of protein size (~100-400 aa) and the number of starting segments (2 to 9). The reason for each protein's difficulty rating varies, but in each case, challenges are reflected in the Max Path score. Any single V ligation plus desulfurization contributes a path length of +4, and thus two 2-segment brackets using a single V site make it into this category, either because the V is paired with a poor thioester (L24) or requires internal C-Acm protection (S17). Other brackets contain poor thioesters at especially difficult locations, either at the only available C site that must be used for the final ligation (IF-3), or adjacent to other poor-thioester sites leading to sequential poor-yielding ligations (L1, L13, S3). Additional brackets contain similar challenges as described for the Medium category, but in higher frequency (L2, S4, S8, EF-Ts, RF1, RRF).