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. 2023 Dec 1;79(Pt 12):1079-1093.
doi: 10.1107/S2059798323008914. Epub 2023 Nov 9.

Improved joint X-ray and neutron refinement procedure in Phenix

Affiliations

Improved joint X-ray and neutron refinement procedure in Phenix

Dorothee Liebschner et al. Acta Crystallogr D Struct Biol. .

Abstract

Neutron diffraction is one of the three crystallographic techniques (X-ray, neutron and electron diffraction) used to determine the atomic structures of molecules. Its particular strengths derive from the fact that H (and D) atoms are strong neutron scatterers, meaning that their positions, and thus protonation states, can be derived from crystallographic maps. However, because of technical limitations and experimental obstacles, the quality of neutron diffraction data is typically much poorer (completeness, resolution and signal to noise) than that of X-ray diffraction data for the same sample. Further, refinement is more complex as it usually requires additional parameters to describe the H (and D) atoms. The increase in the number of parameters may be mitigated by using the `riding hydrogen' refinement strategy, in which the positions of H atoms without a rotational degree of freedom are inferred from their neighboring heavy atoms. However, this does not address the issues related to poor data quality. Therefore, neutron structure determination often relies on the presence of an X-ray data set for joint X-ray and neutron (XN) refinement. In this approach, the X-ray data serve to compensate for the deficiencies of the neutron diffraction data by refining one model simultaneously against the X-ray and neutron data sets. To be applicable, it is assumed that both data sets are highly isomorphous, and preferably collected from the same crystals and at the same temperature. However, the approach has a number of limitations that are discussed in this work by comparing four separately re-refined neutron models. To address the limitations, a new method for joint XN refinement is introduced that optimizes two different models against the different data sets. This approach is tested using neutron models and data deposited in the Protein Data Bank. The efficacy of refining models with H atoms as riding or as individual atoms is also investigated.

Keywords: joint XN refinement; macromolecular crystallography; neutron diffraction.

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Figures

Figure 1
Figure 1
Cumulative number of neutron model depositions in the PDB per year (based on the PDB deposition date).
Figure 2
Figure 2
(a) Histogram of the high-resolution limit for neutron data (retrieved from PDB file header or from primary citations). Bin width 0.2 Å, ticks mark bin limits. (b) High-resolution limit d min of the neutron data against that of the concomitant X-ray data for joint XN entries. The dashed line represents the bisector.
Figure 3
Figure 3
(a) Histogram of neutron data completeness calculated from the deposited data. Bin width 10%, ticks mark bin limits. (b) Completeness of the neutron data against that of the concomitant X-ray data for joint XN entries. The dashed line represents the bisector.
Figure 4
Figure 4
Histogram of the number of unique models determined by neutron diffraction.
Figure 5
Figure 5
Examples of different numbers of alternative conformations and how an asparagine residue appears in electron-density and nuclear scattering length density maps. In all figures gold mesh represents neutron scattering length density and teal mesh represents electron density. Green/red represents mF obsDF model density contoured at 3.0/−3.0 r.m.s.d., carved at 3 Å. (a, b) Ile52 in PDB entry 6l46. Note that there are no notable peaks in the difference density maps around this residue. (c, d) Met266 in PDB entry 6l46. (e, f) Ser160 in PDB entry 3x2o. (g, h) Asn38 in PDB entry 3x2o. The 2mF obsDF model density is contoured at 0.8 r.m.s.d. in (a), (b), (c) and (d), 1 r.m.s.d. in (e) and (f) and 2 r.m.s.d. in (g) and (h). Side-chain H/D atoms are not shown for clarity, except for the Asn group in (h).
Figure 6
Figure 6
Example of differing positions of water molecules. Water molecules in X-ray (teal) and neutron (gold) models of entry 4ny6 are shown. The 2mF obs − DF model density is contoured at 0.8 r.m.s.d.; gold mesh, neutron scattering length density; teal surface, electron density.
Figure 7
Figure 7
Histograms of isotropic ADPs in (a) PDB entry 3x2o and (b) PDB entry 7az3.
Figure 8
Figure 8
(a) R work, (b) R free and (c) R gap for refinements against neutron data only using either the riding or the individual option for H/D atoms. The points are colored according to properties of the data or model, according to the property that showed the most correlation. The gray dashed line represents the bisector.
Figure 9
Figure 9
(a) Comparison of R gap with and without weight optimization for the riding model. (b) R gap for refinements using either the riding or the individual option and with weight optimization for both.
Figure 10
Figure 10
(a) Neutron R work, (b) R free and (c) R gap for refinements of models perturbed at 0.5 Å against neutron data only or using the new joint XN approach. The points are colored according to the high-resolution limit of the neutron data. The gray dashed line represents the bisector. The separate cluster of points (also present in Fig. 11 ▸) corresponds to perturbations of one particular model for which manual refinement and curation may be required to produce better refinement outcomes.
Figure 11
Figure 11
(a) Neutron R work, (b) R free and (c) R gap for refinements of models perturbed at 0.9 Å against neutron data only or using the new joint XN approach. The points are colored according to the high-resolution limit of the neutron data. The gray dashed line represents the bisector.
Figure 12
Figure 12
X-ray (a) R work and (b) R free recomputed from deposited neutron models against refinements using the new joint XN approach (the starting model was not perturbed). The gray dashed line represents the bisector.
Figure 13
Figure 13
Histogram of the percentage of lone waters (water molecules without an equivalent) for the neutron and X-ray models after the new joint XN refinement. (a) Refinements from 0.9 Å perturbations. (b) Refinements from 0.5 Å perturbations. Some models had no waters placed by the ordered solvent procedure in the new joint XN refinement procedure. These correspond to the bar in the 0–10% bin.

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