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Xlink Analyzer: software for analysis and visualization of cross-linking data in the context of three-dimensional structures.

Kosinski J, von Appen A, Ori A, Karius K, Müller CW, Beck M - J. Struct. Biol. (2015)

Bottom Line: Structural characterization of large multi-subunit protein complexes often requires integrating various experimental techniques.To fully adapt XL-MS as a structure characterization technique, we developed Xlink Analyzer, a software tool for visualization and analysis of XL-MS data in the context of the three-dimensional structures.We demonstrate these features by mapping interaction sites within RNA polymerase I and the Rvb1/2 complex.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstraße 1, 69117 Heidelberg, Germany.

No MeSH data available.


Related in: MedlinePlus

Comparison of different experimental cross-linking conditions. (A) Number of cross-links and mono-links with ld-score higher than 30 in each experimental condition. (B) Inter-protein cross-links of ABCα subunit of Pol I (magenta) obtained using a 2 mM and 10 mM concentration of the cross-linker. (C) Cross-links involving the A12 subunit (yellow) as obtained in ‘interval’ mode as compared to the 2 mM cross-linker condition.
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f0025: Comparison of different experimental cross-linking conditions. (A) Number of cross-links and mono-links with ld-score higher than 30 in each experimental condition. (B) Inter-protein cross-links of ABCα subunit of Pol I (magenta) obtained using a 2 mM and 10 mM concentration of the cross-linker. (C) Cross-links involving the A12 subunit (yellow) as obtained in ‘interval’ mode as compared to the 2 mM cross-linker condition.

Mentions: In order to assess the similarity between alternative experimental conditions or biological states and conformations (e.g. a nucleic acid-binding protein complex in the presence or absence of DNA) it is often necessary to compare different XL-MS data sets. Xlink Analyzer allows importing several cross-linking datasets simultaneously to analyze them in combination or separately. To demonstrate this feature, we compared cross-links of Pol I that were obtained using various different chemical reaction conditions using DSS cross-linker. In particular, the cross-linker concentration was varied from 0.05 to 10 mM and the timing of the reaction was such that either all cross-linker was added at once or, alternatively, it was added in several consecutive intervals (see Section 5) in smaller amounts. The ‘interval setting’ involves adding the cross-linker stepwise up to a given concentration, e.g., in 10 steps, each step increasing the concentration by 0.2 mM up to final concentration of 2 mM. This setting is useful for samples with limited availability of which the optimal cross-linker concentration is not known. Although this data set is not quantitative as compared to standards established for conventional shotgun proteomics, namely such that peptide abundance ratios can be calculated, a trend towards more identified mono-links vs cross-links with increasing cross-linker concentrations was apparent (Fig. 5A). Through analysis with Xlink Analyzer, we found that, in this particular data set, different cross-linker concentrations lead to the identification of specific cross-linked residue pairs that otherwise remained undiscovered (see also Fig. A.2). Generally, we could not find a correlation between cross-link-observability in a given condition with structural properties such as solvent accessibility of cross-linked lysine residues (Fig. A.3). Nevertheless, several regions of Pol I were cross-linked only in some of the conditions. For example, no inter-protein cross-link with ld-score larger than 30 was observed for subunit ABCα when 2 mM of cross-linker was used, but two inter-cross-links from ABCα were obtained at 10 mM cross-linker concentration (Fig. 5B). Similarly, no cross-link was observed for linker region of A12 subunit at 2 mM cross-linker concentration but three cross-links were identified when 2 mM cross-linker (total) was added in the ‘interval setting’ mode (Fig. 5C). We thus conclude that Xlink Analyzer can assist experimentalists with the empirical optimization of cross-linking experiments.


Xlink Analyzer: software for analysis and visualization of cross-linking data in the context of three-dimensional structures.

Kosinski J, von Appen A, Ori A, Karius K, Müller CW, Beck M - J. Struct. Biol. (2015)

Comparison of different experimental cross-linking conditions. (A) Number of cross-links and mono-links with ld-score higher than 30 in each experimental condition. (B) Inter-protein cross-links of ABCα subunit of Pol I (magenta) obtained using a 2 mM and 10 mM concentration of the cross-linker. (C) Cross-links involving the A12 subunit (yellow) as obtained in ‘interval’ mode as compared to the 2 mM cross-linker condition.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359615&req=5

f0025: Comparison of different experimental cross-linking conditions. (A) Number of cross-links and mono-links with ld-score higher than 30 in each experimental condition. (B) Inter-protein cross-links of ABCα subunit of Pol I (magenta) obtained using a 2 mM and 10 mM concentration of the cross-linker. (C) Cross-links involving the A12 subunit (yellow) as obtained in ‘interval’ mode as compared to the 2 mM cross-linker condition.
Mentions: In order to assess the similarity between alternative experimental conditions or biological states and conformations (e.g. a nucleic acid-binding protein complex in the presence or absence of DNA) it is often necessary to compare different XL-MS data sets. Xlink Analyzer allows importing several cross-linking datasets simultaneously to analyze them in combination or separately. To demonstrate this feature, we compared cross-links of Pol I that were obtained using various different chemical reaction conditions using DSS cross-linker. In particular, the cross-linker concentration was varied from 0.05 to 10 mM and the timing of the reaction was such that either all cross-linker was added at once or, alternatively, it was added in several consecutive intervals (see Section 5) in smaller amounts. The ‘interval setting’ involves adding the cross-linker stepwise up to a given concentration, e.g., in 10 steps, each step increasing the concentration by 0.2 mM up to final concentration of 2 mM. This setting is useful for samples with limited availability of which the optimal cross-linker concentration is not known. Although this data set is not quantitative as compared to standards established for conventional shotgun proteomics, namely such that peptide abundance ratios can be calculated, a trend towards more identified mono-links vs cross-links with increasing cross-linker concentrations was apparent (Fig. 5A). Through analysis with Xlink Analyzer, we found that, in this particular data set, different cross-linker concentrations lead to the identification of specific cross-linked residue pairs that otherwise remained undiscovered (see also Fig. A.2). Generally, we could not find a correlation between cross-link-observability in a given condition with structural properties such as solvent accessibility of cross-linked lysine residues (Fig. A.3). Nevertheless, several regions of Pol I were cross-linked only in some of the conditions. For example, no inter-protein cross-link with ld-score larger than 30 was observed for subunit ABCα when 2 mM of cross-linker was used, but two inter-cross-links from ABCα were obtained at 10 mM cross-linker concentration (Fig. 5B). Similarly, no cross-link was observed for linker region of A12 subunit at 2 mM cross-linker concentration but three cross-links were identified when 2 mM cross-linker (total) was added in the ‘interval setting’ mode (Fig. 5C). We thus conclude that Xlink Analyzer can assist experimentalists with the empirical optimization of cross-linking experiments.

Bottom Line: Structural characterization of large multi-subunit protein complexes often requires integrating various experimental techniques.To fully adapt XL-MS as a structure characterization technique, we developed Xlink Analyzer, a software tool for visualization and analysis of XL-MS data in the context of the three-dimensional structures.We demonstrate these features by mapping interaction sites within RNA polymerase I and the Rvb1/2 complex.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstraße 1, 69117 Heidelberg, Germany.

No MeSH data available.


Related in: MedlinePlus