(A) Distributions along Cα distance 285–285: the probability of formation of the active C-loop conformation (thick black curve with circles); the probability of finding a chain with the uncharged N terminus due to some appended amino acids (solid brown curve with diamonds); the probability of finding a chain with HB 140-1 (broken blue curve with squares); the probability of finding a chain with HB 214-2 (dotted blue curve with triangles). The total number of chains at each interval of distance 285–285 is, in ascending order, 59, 57, 32, 78, 13, and 15. (B) As in (A), but for ligand-free chains with natively charged N-termini without an appended amino acid. The intervals of 8.5–9.5 and >9.5 are not presented because the number of chains in these intervals are not sufficient to calculate statistics. The total number of chains at each interval of distance 285–285 is, in ascending order, 18, 18, 6, and 16. (C) Two 3CLpro structures that explain the scenario for the accumulation of chains with uncharged N-termini at large distance 285–285, drawn after superposition at the core region. These structures are PDB:6lu7 (SARS-CoV-2): A chain (green) and B chain (cyan); distance 285–285 = 5.314 Å, and PDB:7kfi (SARS-CoV-2) having A chain (salmon) and B chain (yellow) with transparency; distance = 9.858 Å. Structures are superimposed at the core region of 6lu7A and 7kfiB. Only the key parts are illustrated. The entry 6lu7 has a natively charged N terminus, whereas 7kfi has an appended sequence at the N terminus (Gly(-2), Ala(-1), and Met0) drawn as lines. 6lu7 has both HB 140-1 and HB 214-2 (red broken lines). However, 7kfi does not have either HB 140-1 or HB 214-2 and the C-loop is collapsed (Phe140 is oriented to the other direction) because of the uncharged N terminus. Flexible Ser1 of 7kfi induces a shift of the position of Gly2 to break HB 214-2. The absence of HB214-2 causes the motion of domain III to separate Ala285. The upper right diagram shows a schematic diagram of the coupling between the C-loop and domain III via HB 140-1 and HB 214-2.