Preliminary neutron crystallographic analysis of selectively CH₃-protonated deuterated rubredoxin from Pyrococcus furiosus

Neutron crystallography is used to locate H atoms in biological materials and can distinguish between negatively scattering hydrogen-substituted and positively scattering deuterium-substituted positions in isomorphous neutron structures. Recently, Hauptman & Langs (2003; Acta Cryst. A59, 250–254) have shown that neutron diffraction data can be used to solve macromolecular structures by direct methods and that solution is aided by the presence of negatively scattering H atoms in the structure. Selective-labeling protocols allow the design and production of H/D-labeled macromolecular structures in which the ratio of H to D atoms can be precisely controlled. Methyl selective-labeling protocols were applied to introduce (¹H-δ methyl)-leucine and (¹H-γ methyl)-valine into deuterated rubredoxin from Pyrococcus furiosus (PfRd). Here, the production, crystallization and preliminary neutron analysis of a selectively CH₃-protonated deuterated PfRd sample, which provided a high-quality neutron data set that extended to 1.75 Å resolution using the new LADI-III instrument at the Institut Laue-Langevin, are reported. Preliminary analysis of neutron density maps allows unambiguous assignment of the positions of H atoms at the methyl groups of the valine and leucine residues in the otherwise deuterated rubredoxin structure.