Neutron crystallographic refinement with REFMAC 5 of the CCP 4 suite

Hydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand recognition processes, and protein-protein interactions. However, their direct visualisation by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localisation of the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher), that is not often attainable. Differently, neutron diffraction methods enable the visualisation of most H atoms, typically in the form of deuterium (D) atoms at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought. REFMAC 5 of the Collaborative Computational Project No. 4 ( CCP 4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP 4 Monomer Library, the source of prior chemical information used in refinement. One new feature for neutron data analysis in REFMAC 5 is the refinement of the protium/deuterium ( ¹ H/D) fraction. This parameter describes the relative ¹ H/D contribution to neutron scattering for H atoms. The newly developed REFMAC5 algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) by using either ( i ) neutron data-only or ( ii ) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement with REFMAC5 afforded models characterised by R -factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy especially for structures at medium-low resolution.