2. Folding of thermophilic C-domain I m R g = 46 Å N R g = 38 Å I m state never populates I m state never populates I t R g = 60 Å On pathway folding On pathway folding

3. R g = 60 Å

40. Mg 2+ conc. reaches K Mg (I m -to-N) first R g = 60 Å 99.98% 0.01% Neither I m nor N populates yet

72. 0.1% R g = 60 ÅR g = 46 Å R g = 38 Å 10% 90% N starts accumulating, I t still dominants I t is the reference state for the stability of N I m is not important for the stability of N

78. R g = 60 Å R g = 38 Å 50% Mg 2+ conc. reaches K Mg (functional) 49.5% ~ K Mg (I t -to-N) 0.5% I m is negligible

92. R g = 60 Å R g = 38 Å 96% N-state dominants 3.4% I t is the reference state 0.6% I m is negligible

101. R g = 38 Å 99.2% 0.4% When Mg 2+ reaches K Mg (I t -to-I m ), N-state is over 99%

119. R g = 38 Å 99.91% R g = 60 ÅR g = 46 Å 0.08% 0.006% At high enough Mg 2+, I m becomes more stable than I t But none of them populates