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Comparative Study
. 2009 Jun;65(Pt 6):567-73.
doi: 10.1107/S0907444909011548. Epub 2009 May 15.

Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules

Paul D Adams  1 , Marat Mustyakimov, Pavel V Afonine, Paul Langan
Affiliations
Comparative Study

Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules

Paul D Adams et al. Acta Crystallogr D Biol Crystallogr. 2009 Jun.

Abstract

X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallographic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains.

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Figures

Figure 1
Figure 1
General strategy for neutron crystallographic refinement of biological macromolecules. The X-ray model serves as a starting point; H atoms are subsequently added and the model is optimized to obtain the best agreement with the observed neutron data. The initial positions of some H atoms can be predicted from the known geometries of certain functional groups. For others, particularly those in solvent molecules, initial positions can only be determined from inspecting the neutron scattering density map. The proportions of hydrogen and deuterium at labile sites must also be refined in cases where the crystal has been grown or soaked in D2O to enhance its neutron scattering properties. D atoms (neutron scattering length of 6.67 × 10−15 m) appear as strong positive peaks in neutron scattering density maps, thereby revealing the location of isotopically exchanged H atoms and enhancing the scattering power of water molecules, whilst H atoms themselves (neutron scattering length of −3.7 × 10−15 m) appear as negative troughs. For amino-acid side chains that can be ionized, the neutron scattering density maps must be inspected to determine whether or not a H or D atom is present and in what position.
Figure 2
Figure 2
The behavior of crystallographic R factors during refinement of the W3Y rubredoxin mutant. The starting model has been distorted from the published model (r.m.s. coordinate error of 0.5 Å). The values of n R free (a) and x R free (b) are shown as a function of the number of cycles of refinement, where refinement was with either the X-ray data (green) or the neutron data (blue) alone or using both X-ray and neutron data (red). Also shown is the difference n R free − n R work (c) for refinement with either neutron data (blue) alone or using both X-ray and neutron data (red) and the difference x R free − x R work (d) for refinement with either X-ray data (green) alone or using both X-ray and neutron data (red). Some upward deviations in those plots are because new values of ωx and ωn were recalculated after each macrocycle.
Figure 3
Figure 3
The practical benefits of XN refinement. Selected views of superimposed 2mF o − DF c neutron scattering (blue) and electron (green) density maps calculated from the XN structure of diisopropyl fluorophosphate (DFPase; resolutions: X-ray, 1.8 Å; neutron, 2.2 Å; contoured at 1.6σ). In (a) the Nδ2 group of the Asn120 side chain scatters neutrons more strongly than X-rays, making it more easily and accurately orientated. In (b) note that the terminal amine group of the Lys151 side chain has strong neutron scattering density, whereas the methylene groups of the chain have strong electron density. In (c) the orientation of the hydroxyl group of the side chain of Thr102 is clearly indicated by the neutron scattering density. In (d)–(f) D2O molecules appear as either triangles (d), ellipsoids (e) or spheres (f) in the water structure around DFPase. The superposition of the electron-density maps provides complementary information on the location of the O atoms, greatly aiding in the interpretation of these features. In (d) the water molecule is ordered and all three atoms are seen. In (e) the O—D bond is visible with the second D invisible because of rotational disorder around this bond. In (f) the water molecules are completely rotationally disordered.
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References

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