| 1. |
- Guelker, Megan, et al.
(författare)
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Pseudosymmetry, high copy number and twinning complicate the structure determination of Desulfovibrio desulfuricans (ATCC 29577) flavodoxin
- 2009
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Ingår i: Acta crystallographica. Section D, Biological crystallography. - 1399-0047. ; 65:Pt 6, s. 523-34
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of oxidized flavodoxin from Desulfovibrio desulfuricans (ATCC 29577) was determined by molecular replacement in two crystal forms, P3(1)21 and P4(3), at 2.5 and 2.0 A resolution, respectively. Structure determination in space group P3(1)21 was challenging owing to the presence of pseudo-translational symmetry and a high copy number in the asymmetric unit (8). Initial phasing attempts in space group P3(1)21 by molecular replacement using a poor search model (46% identity) and multi-wavelength anomalous dispersion were unsuccessful. It was necessary to solve the structure in a second crystal form, space group P4(3), which was characterized by almost perfect twinning, in order to obtain a suitable search model for molecular replacement. This search model with complementary approaches to molecular replacement utilizing the pseudo-translational symmetry operators determined by analysis of the native Patterson map facilitated the selection and manual placement of molecules to generate an initial solution in the P3(1)21 crystal form. During the early stages of refinement, application of the appropriate twin law, (-h, -k, l), was required to converge to reasonable R-factor values despite the fact that in the final analysis the data were untwinned and the twin law could subsequently be removed. The approaches used in structure determination and refinement may be applicable to other crystal structures characterized by these complicating factors. The refined model shows flexibility of the flavin mononucleotide coordinating loops indicated by the isolation of two loop conformations and provides a starting point for the elucidation of the mechanism used for protein-partner recognition.
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| 2. |
- Meyer, Peter A., et al.
(författare)
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Data publication with the structural biology data grid supports live analysis
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data. sbgrid. org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.
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| 3. |
- Miller, Corwin, et al.
(författare)
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Experimental evolution of adenylate kinase reveals contrasting strategies toward protein thermostability
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 99:3, s. 887-896
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Tidskriftsartikel (refereegranskat)abstract
- Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK(BSUB) Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK(BSUB) G213E decreases protein stability by >25 degrees C, the same mutation in the background of AK(BSUB) Q199R increases stability by 3.4 degrees C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK(BSUB) double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.
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