and PROPKA (version 3.4.0) were used to suggest the protonation state of all residues. Consistent with the literature for the apo protein, 50 a large pKa shift was predicted in both cases for the N-terminal proline residue which is present in the binding site, which produces the neutral form at a pH of 7. The suggested protonation sites for all histidines were taken from H++. Hydrogens were added to MIF using H++ and a hydrogen ion was removed from the N of each of the protonated terminal prolines so as to maintain the hydrogen bond to Tyr36. Based on the results of Qian et al., the neutrality of the terminal proline was maintained in the complex.