Combining X-ray and neutron crystallography with spectroscopy

Abstract

X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H⁺ ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction.

Keywords: neutron protein crystallography; photoreduction; single-crystal spectroscopy.

Figure 1

Absence of evidence is not evidence of absence. 0.9 Å resolution X-ray structure of a phenylalanine residue in PETN reductase (model and data from PDB entry 1vyr; Barna et al., 2001 ▸) showing that even though some H atoms can be seen, those that are known to be present are not always observed (at one meta and the para positions in this case). The electron density is calculated excluding H atoms, the pink density is 2F _o − F _c density contoured at 3.0σ and the green density which shows both H atoms at the ortho position but only one at the meta position is F _o − F _c density contoured at 3.0σ. H atoms are shown in green.

Figure 2

Comparison of calculated nuclear and electron densities at 2.0 Å resolution. Nuclear and electron densities are shown in cyan and magenta, respectively. (a) Nuclear and electron density for a tautomer of neutral histidine where N^δ1 is deuterated. The negative scattering of the two H atoms bonded to the C^β of the side chain has cancelled out the nuclear density at this point. (b) Nuclear and electron density for a tautomer of positively charged histidine where both N^δ1 and N^∊2 are deuterated. (c) Nuclear and electron density calculated for a water molecule. All densities are contoured at 2σ. The densities were calculated in PHENIX (Adams et al., 2009 ▸). H and D atoms are shown in green and white, respectively.

Figure 3

The effects of photoreduction in PETN reductase. (a) A crystal in the oxidized form, the spectrum and the near-planar electron density of the isoalloxazine ring of the FMN molecule. (b) A crystal that has been reduced by X-ray data collection, the resulting spectrum and the bent isoalloxazine ring of the FMN. 2F _o − F _c electron density contoured at 2σ is shown as a blue mesh. Crystallization conditions are shown in Table 2 ▸, data-collection statistics are shown in Table 3 ▸ and refinement statistics are shown in Table 4 ▸.