Abstract <p>Intermediate states of the complex of the macrophage migration inhibitory factor (MIF) with the covalent inhibitor phenylisothiocyanate (PITC) were studied by X-ray diffraction analysis. It was demonstrated that the covalent modification of the N-terminal proline is preceded by the non-covalent binding of the inhibitor in the previously unknown holding site. The holding site was identified due to the use of short-term soaking of a MIF crystal in a ligand-containing cryo-solution followed by flash freezing in a nitrogen stream to collect the X-ray diffraction data at 100 К. A comparison of this structure with the crystal structure of the pre-modified protein revealed the details of the dynamics of the PITC binding.</p>