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Improving experimental phases for strong reflections prior to density modification.

ABSTRACT: Experimental phasing of diffraction data from macromolecular crystals involves deriving phase probability distributions. These distributions are often bimodal, making their weighted average, the centroid phase, improbable, so that electron-density maps computed using centroid phases are often non-interpretable. Density modification brings in information about the characteristics of electron density in protein crystals. In successful cases, this allows a choice between the modes in the phase probability distributions, and the maps can cross the borderline between non-interpretable and interpretable. Based on the suggestions by Vekhter [Vekhter (2005), Acta Cryst. D61, 899-902], the impact of identifying optimized phases for a small number of strong reflections prior to the density-modification process was investigated while using the centroid phase as a starting point for the remaining reflections. A genetic algorithm was developed that optimizes the quality of such phases using the skewness of the density map as a target function. Phases optimized in this way are then used in density modification. In most of the tests, the resulting maps were of higher quality than maps generated from the original centroid phases. In one of the test cases, the new method sufficiently improved a marginal set of experimental SAD phases to enable successful map interpretation. A computer program, SISA, has been developed to apply this method for phase improvement in macromolecular crystallography.

SUBMITTER: Uervirojnangkoorn M

PROVIDER: S-EPMC3792643 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Improving experimental phases for strong reflections prior to density modification.

Uervirojnangkoorn Monarin M Hilgenfeld Rolf R Terwilliger Thomas C TC Read Randy J RJ

Acta crystallographica. Section D, Biological crystallography 20130920 Pt 10

Experimental phasing of diffraction data from macromolecular crystals involves deriving phase probability distributions. These distributions are often bimodal, making their weighted average, the centroid phase, improbable, so that electron-density maps computed using centroid phases are often non-interpretable. Density modification brings in information about the characteristics of electron density in protein crystals. In successful cases, this allows a choice between the modes in the phase prob ...[more]

PMID: 24100322

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Project description:BackgroundAnimal fracture models, primarily performed in rats, are crucial to investigate normal and pathological bone healing. However, results of biomechanical testing representing a major outcome measure show high standard deviations often precluding statistical significance. Therefore, the aim of our study was a systematical examination of biomechanical characteristics of rat femurs during three-point bending. Furthermore, we tried to reduce variation of results by individually adapting the span of bearing and loading areas to the bone's length.MethodsWe examined 40 paired femurs of male Wistar-rats by DXA (BMD and BMC of the whole femur) and pQCT-scans at the levels of bearing and loading areas of the subsequent biomechanical three-point bending test. Individual adjustment of bearing and loading bars was done respecting the length of each specimen. Subgroups of light (<?400 g, n?=?22) and heavy (>?400 g, n?=?18) animals were formed and analysed separately. We furthermore compared the results of the individualised bending-setting to 20 femurs tested with a fix span of 15 mm.ResultsFemurs showed a length range of 34 to 46 mm. The failure loads ranged from 116 to 251 N (mean 175.4?±?45.2 N; heavy animals mean 221?±?18.9 N; light animals mean 138.1?±?16.4 N) and stiffness ranged from 185 N/mm to 426 N/mm (mean 315.6?±?63 N/mm; heavy animals mean 358.1?±?34.64 N/mm; light animals mean 280.8?±?59.85 N/mm). The correlation of densitometric techniques and failure loads was high (DXA R2?=?0.89 and pQCT R2?=?0.88). In comparison to femurs tested with a fix span, individual adaptation of biomechanical testing homogenized our data significantly. Most notably, the standard deviation of failure loads (221?±?18.95 N individualized setting vs. 205.5?±?30.36 N fixed) and stiffness (358.1?±?34.64 N/mm individualized setting vs. 498.5?±?104.8 N/mm fixed) was reduced by at least one third.ConclusionsTotal variation observed in any trait reflects biological and methodological variation. Precision of the method hence affects the statistical power of the study. By simply adapting the setting of the biomechanical testing, interindividual variation could be reduced, which improves the precision of the method significantly.

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Improving experimental phases for strong reflections prior to density modification.

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Improving experimental phases for strong reflections prior to density modification.

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