High resolution neutron protein crystallography. Hydrogen and hydration in proteins

Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins, and the development of the neutron imaging plate (NIP) became a breakthrough event in neutron protein crystallography. The general features of the NIP are reviewed. A high resolution neutron diffractometer dedicated to biological macromolecules (BIX-3) with the NIP has been constructed at Japan Atomic Energy Research Institute and this has enabled 1.5 Angstrom resolution structural analyses of several proteins to be carried out. The specifications of BIX-3 and LADI (a quasi-Laue type diffractometer installed in the Institut Laue-Langevin) are compared. The crystal structures of myoglobin, wild type rubredoxin and a mutant of rubredoxin have been carried out using BIX-3. From these studies, several topics, such as the location of hydrogen bonds and certain acidic hydrogen atoms, the identification of methyl hydrogen atoms, details of H/D exchange and dynamical behavior of hydration structures have been investigated, and important information has been extracted from the structural results. Finally, a systematic procedure to grow large single crystals of proteins or nucleic acids is described.