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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

Analysis of cross-links mapped to Pol I crystal structure. (A) Overall view of Pol I structure (PDB code: 4C3H) (Fernandez-Tornero et al., 2013) showing cross-links with ld-score 30 or higher. Satisfied cross-links (using a distance threshold of 30 Å) are colored blue, violated cross-links (using 30 Å distance threshold) are colored red. (B) Cross-links suggest two alternative interaction sites for the extended loop of the A190 subunit. Dashed lines indicate regions missing in the structure. For clarity, only A190 and A135 subunits and intra-links of A190 subunit and inter-links between A190 and A135 subunits are displayed. Displaying individual subunits and specific cross-link types is facilitated through appropriate panels in Xlink Analyzer. The extended loop is colored cyan using standard coloring tools of UCSF Chimera. (C) Mapping interactions to the tWH domain that forms an extension of subunit A49 and is disordered in the crystal structure. The tWH domain was defined in the project setup as residues 172–403 and the residues cross-linking to this domain were highlighted using Xlink Analyzer (colored dark red as the color of this domain defined in the setup). The approximate position of A49-tWH based on cross-links is indicated.
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f0010: Analysis of cross-links mapped to Pol I crystal structure. (A) Overall view of Pol I structure (PDB code: 4C3H) (Fernandez-Tornero et al., 2013) showing cross-links with ld-score 30 or higher. Satisfied cross-links (using a distance threshold of 30 Å) are colored blue, violated cross-links (using 30 Å distance threshold) are colored red. (B) Cross-links suggest two alternative interaction sites for the extended loop of the A190 subunit. Dashed lines indicate regions missing in the structure. For clarity, only A190 and A135 subunits and intra-links of A190 subunit and inter-links between A190 and A135 subunits are displayed. Displaying individual subunits and specific cross-link types is facilitated through appropriate panels in Xlink Analyzer. The extended loop is colored cyan using standard coloring tools of UCSF Chimera. (C) Mapping interactions to the tWH domain that forms an extension of subunit A49 and is disordered in the crystal structure. The tWH domain was defined in the project setup as residues 172–403 and the residues cross-linking to this domain were highlighted using Xlink Analyzer (colored dark red as the color of this domain defined in the setup). The approximate position of A49-tWH based on cross-links is indicated.

Mentions: To demonstrate the potential of Xlink Analyzer, we performed XL-MS analysis of RNA Polymerase (Pol) I from Saccharomyces cerevisiae. We purified Pol I as previously described (Fernandez-Tornero et al., 2013) and subjected it to varying concentrations of H12/D12 isotope-coded, di-succinimidyl-suberate (DSS) cross-linker, which reacts with amine groups of lysine residues and protein N-termini (Section 5). The cross-linking reaction was quenched with ammonium bicarbonate and proteins were digested with trypsin. The cross-linked peptides were enriched by size exclusion chromatography as previously described (Leitner et al., 2012) and subjected to LC–MS/MS analysis. The cross-links were then identified from the MS spectra using the xQuest/xProphet (Leitner et al., 2014a). After data import into Xlink Analyzer and stringent filtering according to the cross-link confidence score (xQuest ld-score > 30), we mapped the identified cross-links onto the crystal structure of Pol I (Engel et al., 2013; Fernandez-Tornero et al., 2013)(Fig. 2A). Using Xlink Analyzer, we found 106 unique cross-linked residue pairs that could be mapped to the structure, of which five are violated using 30 Å distance threshold (Merkley et al., 2014). Interestingly, four out of the five violated cross-links originate from an extended loop inside the DNA-binding cleft of Pol I (Fig. 2B). This loop has been suggested to be a mobile regulatory element that becomes ordered only under certain conditions (Engel et al., 2013; Fernandez-Tornero et al., 2013). Several cross-links agree with the position of the extended loop in the Pol I cleft as suggested by the crystal structures, whereas three of the violated cross-links consistently suggest an alternative position on the clamp head domain. Thus, the extended loop likely binds to the clamp head in an alternative conformational state of Pol I or transiently interacts with that region when moving to the DNA-binding cleft.


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)

Analysis of cross-links mapped to Pol I crystal structure. (A) Overall view of Pol I structure (PDB code: 4C3H) (Fernandez-Tornero et al., 2013) showing cross-links with ld-score 30 or higher. Satisfied cross-links (using a distance threshold of 30 Å) are colored blue, violated cross-links (using 30 Å distance threshold) are colored red. (B) Cross-links suggest two alternative interaction sites for the extended loop of the A190 subunit. Dashed lines indicate regions missing in the structure. For clarity, only A190 and A135 subunits and intra-links of A190 subunit and inter-links between A190 and A135 subunits are displayed. Displaying individual subunits and specific cross-link types is facilitated through appropriate panels in Xlink Analyzer. The extended loop is colored cyan using standard coloring tools of UCSF Chimera. (C) Mapping interactions to the tWH domain that forms an extension of subunit A49 and is disordered in the crystal structure. The tWH domain was defined in the project setup as residues 172–403 and the residues cross-linking to this domain were highlighted using Xlink Analyzer (colored dark red as the color of this domain defined in the setup). The approximate position of A49-tWH based on cross-links is indicated.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359615&req=5

f0010: Analysis of cross-links mapped to Pol I crystal structure. (A) Overall view of Pol I structure (PDB code: 4C3H) (Fernandez-Tornero et al., 2013) showing cross-links with ld-score 30 or higher. Satisfied cross-links (using a distance threshold of 30 Å) are colored blue, violated cross-links (using 30 Å distance threshold) are colored red. (B) Cross-links suggest two alternative interaction sites for the extended loop of the A190 subunit. Dashed lines indicate regions missing in the structure. For clarity, only A190 and A135 subunits and intra-links of A190 subunit and inter-links between A190 and A135 subunits are displayed. Displaying individual subunits and specific cross-link types is facilitated through appropriate panels in Xlink Analyzer. The extended loop is colored cyan using standard coloring tools of UCSF Chimera. (C) Mapping interactions to the tWH domain that forms an extension of subunit A49 and is disordered in the crystal structure. The tWH domain was defined in the project setup as residues 172–403 and the residues cross-linking to this domain were highlighted using Xlink Analyzer (colored dark red as the color of this domain defined in the setup). The approximate position of A49-tWH based on cross-links is indicated.
Mentions: To demonstrate the potential of Xlink Analyzer, we performed XL-MS analysis of RNA Polymerase (Pol) I from Saccharomyces cerevisiae. We purified Pol I as previously described (Fernandez-Tornero et al., 2013) and subjected it to varying concentrations of H12/D12 isotope-coded, di-succinimidyl-suberate (DSS) cross-linker, which reacts with amine groups of lysine residues and protein N-termini (Section 5). The cross-linking reaction was quenched with ammonium bicarbonate and proteins were digested with trypsin. The cross-linked peptides were enriched by size exclusion chromatography as previously described (Leitner et al., 2012) and subjected to LC–MS/MS analysis. The cross-links were then identified from the MS spectra using the xQuest/xProphet (Leitner et al., 2014a). After data import into Xlink Analyzer and stringent filtering according to the cross-link confidence score (xQuest ld-score > 30), we mapped the identified cross-links onto the crystal structure of Pol I (Engel et al., 2013; Fernandez-Tornero et al., 2013)(Fig. 2A). Using Xlink Analyzer, we found 106 unique cross-linked residue pairs that could be mapped to the structure, of which five are violated using 30 Å distance threshold (Merkley et al., 2014). Interestingly, four out of the five violated cross-links originate from an extended loop inside the DNA-binding cleft of Pol I (Fig. 2B). This loop has been suggested to be a mobile regulatory element that becomes ordered only under certain conditions (Engel et al., 2013; Fernandez-Tornero et al., 2013). Several cross-links agree with the position of the extended loop in the Pol I cleft as suggested by the crystal structures, whereas three of the violated cross-links consistently suggest an alternative position on the clamp head domain. Thus, the extended loop likely binds to the clamp head in an alternative conformational state of Pol I or transiently interacts with that region when moving to the DNA-binding cleft.

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