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. 2014 May;70(Pt 5):1346-56.
doi: 10.1107/S1399004714003277. Epub 2014 Apr 30.

Flexible torsion-angle noncrystallographic symmetry restraints for improved macromolecular structure refinement

Jeffrey J Headd  1 , Nathaniel Echols  1 , Pavel V Afonine  1 , Nigel W Moriarty  1 , Richard J Gildea  1 , Paul D Adams  1
Affiliations

Flexible torsion-angle noncrystallographic symmetry restraints for improved macromolecular structure refinement

Jeffrey J Headd et al. Acta Crystallogr D Biol Crystallogr. 2014 May.

Abstract

One of the great challenges in refining macromolecular crystal structures is a low data-to-parameter ratio. Historically, knowledge from chemistry has been used to help to improve this ratio. When a macromolecule crystallizes with more than one copy in the asymmetric unit, the noncrystallographic symmetry relationships can be exploited to provide additional restraints when refining the working model. However, although globally similar, NCS-related chains often have local differences. To allow for local differences between NCS-related molecules, flexible torsion-based NCS restraints have been introduced, coupled with intelligent rotamer handling for protein chains, and are available in phenix.refine for refinement of models at all resolutions.

Keywords: NCS; automation; macromolecular crystallography; noncrystallographic symmetry; refinement.

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Figures

Figure 1
Figure 1
Flow diagram of NCS-related automated rotamer correction.
Figure 2
Figure 2
NCS rotamer consistency correction for IleA120 in PDB entry 1m0o at 1.7 Å resolution. (a) Starting orientation of IleA120 in 1m0o in the tt rotamer. Bad steric clashes (>0.4 Å) are depicted in hot pink. (b) χ angles adjusted to match the pt rotamer orientation of the NCS-related IleB120 side chain. (c) Result of the local conformation search, including backrub motion, shown in green. (d) Following a default run of phenix.refine, the correct pt rotamer is fitted to the density map. All maps are 2mF o − DF c maps contoured at 1.2σ. Images were generated using KiNG (Chen et al., 2009 ▶).
Figure 3
Figure 3
Comparison of Arg63 of PDB entry 1sar following molecular replacement plus AutoBuild with refinement in phenix.refine. (a) Arg63 from the A chain. The final position is shown for 1sar (dark blue), refinement with torsion NCS restraints (light blue), refinement without NCS restraints (green) and refinement with global NCS restraints (hot pink), with rotamer states indicated in matching colors. (b) Arg63 from the A chain. The final position is shown for PDB entry 1sar (dark blue), refinement with torsion NCS restraints (light blue), refinement without NCS restraints (green) and refinement with global NCS restraints (hot pink), with rotamer states indicated in matching colors. All maps are 2mF o − DF c maps contoured at 1σ. Images were generated using KiNG (Chen et al., 2009 ▶).
Figure 4
Figure 4
Comparison of the Arg63 loop in the A and B chains in the 1.8 Å resolution RNAse S structure (PDB entry 1sar). (a) Overlay of the Arg63 loop region from the A chain (blue) and B chain (green), illustrating the rotameric difference of Arg63 between the chains. (b) Arg63 from the A chain with 2mF o − DF c density map. (c) Arg63 from chain B with 2mF o − DF c density map. All maps are contoured at 1σ. Images were generated using KiNG (Chen et al., 2009 ▶).
Figure 5
Figure 5
(a) Plot of residual R free values for refinements of a set of 56 moderate-resolution structures using torsion NCS with rotamer correction (blue diamonds), global NCS (red squares) and local NCS restraints in REFMAC5 (Murshudov et al., 2011; green squares). The residual R free is calculated as R free(NCS) − R free(no NCS). (b) Plot of residual R free values for refinements using torsion NCS restraints only (purple crosses), rotamer correction only (light blue dashed crosses) and torsion NCS with rotamer correction (blue diamonds). Data for both plots are plotted on the x axis in order of increasing residual R free for refinement with torsion NCS with rotamer correction in phenix.refine.
Figure 6
Figure 6
(a) Rotamer outlier percentage analysis for a set of 56 test refinements using torsion NCS with rotamer correction (blue diamonds), global NCS (red triangles), no NCS (orange circles) and REFMAC5 (green triangles). (b) Rotamer outlier percentage for torsion NCS restraints only (purple crosses), rotamer correction only (light blue dashed crosses) and torsion NCS with rotamer correction (blue diamonds). Both plots are sorted by increasing rotamer outlier percentage using torsion NCS restraints with rotamer correction.
Figure 7
Figure 7
Comparison of NCS-related LeuA88 and LeuB88 of PDB entry 1jbb refined with and without torsion NCS restraints. (a) Refinement without NCS restraints allows the incorrectly built LeuB88 side chain to remain a tp rotamer while distorting the surrounding backbone geometry. (b) Refinement using torsion NCS restraints preserves similar backbone geometry, which causes the incorrectly built LeuB88 side chain to present as an outlier. Images were generated using KiNG (Chen et al., 2009 ▶).
Figure 8
Figure 8
(a) Clashscore (Chen et al., 2010 ▶) analysis for a set of 56 test refinements using torsion NCS with rotamer correction (blue diamonds), global NCS (red triangles), no NCS (orange circles) and REFMAC5 (green triangles). (b) Clashscore analysis for torsion NCS restraints only (purple crosses), rotamer correction only (light blue dashed crosses) and torsion NCS with rotamer correction (blue diamonds). Both plots are sorted by increasing rotamer outlier percentage using torsion NCS restraints with rotamer correction.
Figure 9
Figure 9
(a) Ramachandran outlier percentage analysis for a set of 56 test refinements using torsion NCS with rotamer correction (blue diamonds), global NCS (red triangles), no NCS (orange circles) and REFMAC5 (green triangles). (b) Ramachandran outlier percentage for torsion NCS restraints only (purple crosses), rotamer correction only (light blue dashed crosses) and torsion NCS with rotamer correction (blue diamonds). Both plots are sorted by increasing Ramachandran outlier percentage using torsion NCS restraints with rotamer correction.
Figure 10
Figure 10
Handling of LeuB88 in the refinement of PDB entry 1jbb (2.0 A resolution). (a) Following ten macro-cycles of phenix.refine, LeuB88 refines to an incorrect tp rotamer (no NCS restraints, shown in green) or an outlier (torsion NCS restraints, shown in pink). Simple rotation to a correct mt rotamer (purple) does not improve the density fit sufficiently for acceptance by the rotamer correction routine. (b) Correct placement of the mt rotamer (blue) following ‘autofit best rotamer’ using Coot (Emsley et al., 2010 ▶). (c) Following five additional macro-cycles of phenix.refine using torsion NCS restraints, the correct mt rotamer remains and the positive mF o − DF c density peak is eliminated. 2mF o − DF c maps (gray mesh) are contoured at 1.2σ. mF o − DF c maps (green peak) are contoured at 3.5σ. Images were generated using KiNG (Chen et al., 2009 ▶).
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References

    1. Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352–367. - PMC - PubMed
    1. Bricogne, G. (1976). Acta Cryst. A32, 832–847.
    1. Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O. & Vonrhein, C. (2010). BUSTER. Global Phasing Limited, Cambridge, England.
    1. Brünger, A. T. (1992). Nature (London), 355, 472–475. - PubMed
    1. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. & Warren, G. L. (1998). Acta Cryst. D54, 905–921. - PubMed

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