Quantum refinement with electron diffraction and X-ray free-electron laser data: comparative study of ribonucleotide...

Quantum refinement (QR) is an approach in which the empirical restraints used in standard structural refinement to ensure that the details of the structure, e.g. bond lengths and angles, make chemical sense are replaced by more accurate quantum mechanical calculations for a small but interesting part of the structure. QR has previously been used for X-ray and neutron crystallography, cryogenic electron microscopy, nuclear magnetic resonance, and extended X-ray absorption fine structure. Here, QR is used for the first time for X-ray free-electron laser (XFEL) crystallography and microcrystal electron diffraction (MicroED). As a test case, we use six structures of the R2a protein of ribonucleotide reductase, concentrating on the binuclear Fe₂ site in either the oxidized (Fe₂ ^III) or reduced (Fe₂ ^II) state, two each from single-crystal X-ray (SCX) crystallography, XFEL crystallography or MicroED. The results show that QR works well for data from all three radiation sources, even though scattering factors for neutral atoms had to be used for MicroED. QR corrects unrealistically short Fe-O distances in the reduced SCX structure and gives improved real-space Z scores for the reduced MicroED structure. The three methods give similar structures, apart from variation in the weak water ligands and in the binding of carboxylate groups (monodentate, bidentate or a mixture). By performing QR for three protonation states of the bridging solvent molecule, we could show that it is undoubtedly a water molecule in the reduced XFEL and MicroED structures (it is not present in the SCX structure) and that it is not water in the oxidized structures. The XFEL data indicate that it is O^2- in the oxidized XFEL structure, in agreement with the spectroscopic results. However, for the SCX structure, O^2- and OH^- give comparable results, whereas OH^- is slightly preferred in the MicroED structure. This indicates that the SCX and MicroED structures may be partly photoreduced during data collection.