Neutron crystallography: opportunities, challenges, and limitations

Abstract

Neutron crystallography has had an important, but relatively small role in structural biology over the years. In this review of recently determined neutron structures, a theme emerges of a field currently expanding beyond its traditional boundaries, to address larger and more complex problems, with smaller samples and shorter data collection times, and employing more sophisticated structure determination and refinement methods. The origin of this transformation can be found in a number of advances including first, the development of neutron image-plates and quasi-Laue methods at nuclear reactor neutron sources and the development of time-of-flight Laue methods and electronic detectors at spallation neutron sources; second, new facilities and methods for sample perdeuteration and crystallization; third, new approaches and computational tools for structure determination.

Figure 1

(a) A 2Fo-Fc nuclear density map calculated from the joint refined X-ray (1.0Å) and neutron (1.8Å) structure of the enzyme endothiapepsin clearly shows the protonation state of the enzyme and the bound gem-diol inhibitor PD-135,040 in the active site [10,11]. (b) A topological representation of the extent of H/D exchange at backbone amide groups in the enzyme diisopropyl fluorophosphatase [9•]: blue, nonexchanged; red, fully exchanged. The extent of isotopic exchange is a measure of solvent accessibility and local refolding dynamics. (c) A 2Fo-Fc nuclear density map calculated from the refined 1.6Å neutron structure of a rubredoxin triple mutant, showing a view of residue Trp36. Blue and red contours show positive and negative densities, respectively. The green broken line shows a hydrogen bond between the N—D bond of Trp36 and the carboxyl group of Glu18. Note the positive contours of the N—D group and the entire D₂O molecule, as opposed to the negative contours of the H atoms of the C—H bonds [43]. (d) Superimposed 2Fo-Fc nuclear (green) and electron (red) density maps calculated from the joint refined X-ray (1.1Å) and neutron (2.5Å) structure of photoactive yellow protein, showing residues Asn13. Note the Nδ2 group of Asn13 scatters neutrons more strongly than X-rays and makes the determination of the orientation of Asn side chains much easier and more accurate [16••]. (e) Superimposed 2Fo-Fc nuclear (blue) and electron (red) density maps calculated from the joint refined X-ray (1.8Å) and neutron (2.2Å) structure of enzyme diisopropyl fluorophosphatase, showing the active site. Note that water molecules are clearly oriented in the joint structure [9]. (f) A 2Fo-Fc nuclear density map calculated from the refined 1.8Å neutron structure of the enzyme D-xylose isomerase, showing a view of residue His54. Although the protonation state of this residue is ambiguous in the atomic resolution (0.94Å) X-ray structure, it is clear from the nuclear density that both Nδ1 and Nε2 are protonated [6••].

Figure 2

Superposition of catalytic residues Lys77 and Asp43 from a fully deuterated aldose reductase model (jointly refined against room temperature X + N diffraction data) with a room temperature neutron (2.2Å resolution) 2Fo-Fc map (magenta contours at 2σ) and a 100 K X-ray high-resolution (0.8Å resolution) Fo-Fc map (blue contours at 3σ). Both maps indicate that Lys77 exists in a neutral (doubly protonated) conformation, shown in the model. This neutral conformation, unexpected for a lysine residue hydrogen bonded to an aspartate, led to the proposal of a new catalytic mechanism [4••]. Note that useful neutron diffraction data could be obtained from a crystal with a volume of 0.15 mm³, radically smaller than usual for neutron diffraction studies.