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xVis: a web server for the schematic visualization and interpretation of crosslink-derived spatial restraints

xVis: a web server for the schematic visualization and interpretation of crosslink-derived... W362–W369 Nucleic Acids Research, 2015, Vol. 43, Web Server issue Published online 08 May 2015 doi: 10.1093/nar/gkv463 xVis: a web server for the schematic visualization and interpretation of crosslink-derived spatial restraints 1 1 2 1,* Maximilian Grimm , Tomasz Zimniak , Abdullah Kahraman and Franz Herzog Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universitat ¨ Munchen, ¨ Munich 81377, Germany and Institute of Molecular Life Sciences, University of Zurich, Zurich 8057, Switzerland Received February 25, 2015; Revised April 17, 2015; Accepted April 27, 2015 ABSTRACT only label surface exposed residues but also covalently link residues and thus reveal their proximity within the three- The identification of crosslinks by mass spectrom- dimensional (3D) structure of biomolecules and their com- etry has recently been established as an integral plexes. part of the hybrid structural analysis of protein com- In recent years, advances in mass spectrometric instru- plexes and networks. The crosslinking analysis de- mentation that facilitate the detection of modified pep- termines distance restraints between two covalently tides at high mass accuracy, resolution and sensitivity re- linked amino acids which are typically summarized sulted in the development of different workflows for the identification of crosslinked peptides in complex protein in a table format that precludes the immediate and samples (2,3). Chemical crosslinking combined with mass comprehensive interpretation of the topological data. spectrometry predominantly involves reagents carrying N- xVis displays crosslinks in clear schematic represen- hydroxy-succinimidyl esters which are reactive against pri- tations in form of a circular, bar or network diagram. mary amines of lysine side chains and protein N-termini. The interactive graphs indicate the linkage sites and Chemistries targeting acidic amino acids have been success- identification scores, depict the spatial proximity of fully introduced (4), however, have not yet led to routine ap- structurally and functionally annotated protein re- plications. Similar spatial information is obtained by com- gions and the evolutionary conservation of amino bining mass spectrometry with the site-specific incorpora- acids and facilitate clustering of proteins into sub- tion of photoactivatable amino acids which give rise to rad- complexes according to the crosslink density. Fur- icals highly reactive against various side chains (5). The reported protocols for the identification of thermore, xVis offers two options for the qualitative crosslinked residues differ by the physicochemical prop- assessment of the crosslink identifications by filter- erties of the spacer arm (6) and by the dedicated analysis ing crosslinks according to identification scores or software. In particular, recent workflows implemented false discovery rates and by displaying the corre- isotope-coded and collision-induced-dissociation (CID) sponding fragment ion spectrum of each crosslink cleavable spacer arms. Isotope-tagging introduces an for the manual validation of the mass spectrometric isotopic mass shift which detects crosslinks as isotopic data. Our web server provides an easy-to-use tool pairs and aids in identifying the fragment ions derived from for the fast topological and functional interpretation the two crosslinked peptides by the search engine xQuest of distance information on protein complex architec- (7). CID cleavable crosslinkers generate reporter ions and tures and for the evaluation of crosslink fragment give rise to the linear peptides modified with the remaining ion spectra. xVis is available under a Creative Com- crosslinker masses which facilitates the identification of the mons Attribution-ShareAlike 4.0 International license crosslink sites (8). Regardless of the workflow, the structural informa- at http://xvis.genzentrum.lmu.de/. tion determined by chemical and photoactivatable (9–11) crosslinking is a distance restraint which is based on the INTRODUCTION length of the reagent. We are using disuccinimidyl suberate as a crosslinker which has a spacer arm length of 11.4 A. Chemical labeling of functional groups has a long-standing In our study on a network of modular protein phosphatase history in the structural analysis of proteins and ribonucleic 2A (PP2A) complexes, crosslinks were evaluated by measur- acids (1). Initially, the tagging with different chemistries ing the distances between the two bridged lysines on avail- such as hydrogen/deuterium exchange or hydroxyl radical able X-ray structures and homology models (12). The me- labeling detected solvent accessible residues, also known dian Euclidian C-C distances of 287 intra-protein and as footprinting. In contrast, bifunctional reagents do not To whom correspondence should be addressed. Tel: +49 89 2180 76755; Fax: +49 89 2180 76999; Email: [email protected] C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2015, Vol. 43, Web Server issue W363 70 inter-protein crosslinks were 15.4 and 19.6 A, respec- representation below or above a certain cut-off value. For tively. The recently released Xlink Analyzer software enables crosslink datasets identified by the search engine xQuest, the automatic analysis and visualization of crosslinks in the we offer the possibility to connect to axQuest server for context of the 3D structures (13). In comparison to Euclid- the manual inspection of the crosslink fragment ion spec- ian distances the simulation of distance restraints on the tra. We also implemented sorting algorithms that arrange solvent accessible protein surface has been shown to be su- and group subunits according to crosslink densities. Strik- perior in constraining computational molecular modeling ingly, all subunits of the INO80 - nucleosome complex are and docking experiments (14,15). Inter-protein crosslinks correctly assigned to the previously identified subcomplexes indicate direct protein–protein contacts and will thus sig- solely based on hierarchical clustering or the Markov Clus- nificantly support systems biology efforts in predicting and ter (MCL) Algorithm using crosslink-derived restraints. reconstructing protein interactions of biological networks (16). MATERIALS AND METHODS The potential of crosslinks in integrating structural data xVis setup of different sources has been demonstrated by several groups. In particular, the heterogeneity of the sample prepa- xVis is a server-client-based software solution which uses rations and the intrinsic flexibility of macromolecular pro- PHP to fetch protein information from the InterPro (http:// tein complexes have in many cases prevented their structural www.ebi.ac.uk/interpro/) and UniProt (http://www.uniprot. elucidation by established high-resolution methods. These org/) databases. The protein lengths and secondary struc- limitations led to the design of hybrid approaches that ap- tures from UniProt are accessed with dbfetch (22) provided ply crosslink-derived distance restraints and computational by EMBL-EBI using UniProt identifiers. The annotations modeling to integrate crystal structures and models of in- are downloaded from InterPro with MartWizard (23). The dividual subunits with electron microscopic (EM) densities diagrams are generated on the client by a JavaScript library and subunit localization (17–19). Depending on the input named D3 (Data-Driven Documents) (24). D3 is used to data the integrative structural analysis results in models at generate scalable vector graphics (SVG) and to animate the different levels of detail. In recent studies we have obtained arrangements of proteins in the network diagram with force a hybrid model of RNA polymerase I at near atomic res- layout. To get a similar design on each browser we used olution (20) whereas the availability of only sparse high- Bootstrap and Jasny Bootstrap. resolution structural data on the INO80 complex resulted xVis can be installed on a server with PHP by coping the in a topological model of less detail (21). source code into the web directory of the server. We also The budding yeast INO80 complex is composed of provide a portable version containing the XAMPP server 15 subunits and belongs to the INO80/SWR1 family for Windows. The local installation of xVis is important for chromatin remodelers. Its important role in transcrip- its connection to the xQuest server. The URL of the xQuest tion and genome maintenance is the exchange of histone server has to be entered on the ‘Settings’ page. H2A.Z/H2B dimers with free H2A/H2B. To elucidate the mechanism of histone exchange we analyzed the architec- Input data ture of the INO80 complex interacting with its nucleosome substrate by combining EM 3D densities and subunit lo- The input data for the visualization of crosslink datasets calization with chemical crosslinking and mass spectrome- comprises the coordinates of crosslinks including protein try. This hybrid structural analysis revealed distinct struc- descriptors and positions of the linked residues, protein tural modules of an embryo-shaped complex that embraces lengths information, quality scores for the mass spectro- the nucleosome like a flexible clamp. Chemical crosslinking metric identification, annotation of protein domains and together with subunit localization and biochemical assays xQuest references which provide the possibility to link each identified individual subcomplexes and assigned them to crosslink with the corresponding fragment ion spectrum. structural models (Figure 1A). Furthermore, the crosslink- The input data are uploaded as comma separated files in derived spatial restraints were used to draw a proximity the ‘Input Parameters’ form. In addition to the main input map of structurally and functionally annotated domains file, two accessory files help to customize the representa- which provided detailed insights into the mode of nucleo- tions. xVis identifies the input values by the column head- some binding and histone H2A variant exchange. ings, hence, the order of columns is not important whereas We thus developed xVis, a software solution for the fast the headings have to precisely match the names given in the and schematic visualization and interpretation of distance sample input files which can be retrieved from the ‘Input information on protein complexes and networks. xVis is Parameters’ and ‘Downloads’ page. able to display crosslink-derived distances obtained by dif- The main ‘Crosslink Data’ file is derived from the xQuest ferent workflows in form of a circular, bar or network plot. output file, however, several crosslink datasets obtained by The import of structurally and functionally annotated do- other workflows can be visualized if delivered in the appro- mains as well as the evolutionary conservation rates of priate input format. The ‘Crosslink Data’ file has to provide amino acids is crucial for the initial structural and func- the protein names and the position of the linkage sites and tional interpretation prior to the more laborious genera- optionally, may contain the identification score, the FDR tion of 3D models. Furthermore, the program offers two and the xQuest reference of the fragment ion spectra. The options for the evaluation of crosslink identifications. The xQuest reference of each crosslink is generated from val- filtering of crosslinks according to the identification score ues of different columns, hence requiring a complete xQuest or the false discovery rate (FDR) allows their selective output file as ‘Crosslink Data’ input file. W364 Nucleic Acids Research, 2015, Vol. 43, Web Server issue Figure 1. Topological analysis of the INO80 chromatin remodeler in complex with its nucleosome substrate. (A) Identification of subcomplexes and as- signment to different structural modules using EM, chemical crosslinking and biochemical assays. (B) Crosslinked INO80 subunits are visualized and alphabetically sorted in a circular representation using xVis. The protein names are colored according to the modules in (A). (C) Hierarchical clustering of INO80 subunits based on crosslinks displayed in a circular diagram by xVis.(D) Hierarchical clustering of INO80 subunits based on crosslinks shown in a bar diagram by xVis. Bars represent InterPro domains (Figure 3A) (inter-protein crosslinks in black, intra-protein crosslinks in red). Nucleic Acids Research, 2015, Vol. 43, Web Server issue W365 Figure 2. Topological analysis of the INO80 - nucleosome complex by applying the Markov Cluster (MCL) Algorithm in the network diagram. (A) Subunits non-clustered (all proteins blue colored). (B) Subunits clustered with the MCL parameters expansion=2 and inflation =1 separates the Nhp10 subcomplex (light blue). (C) Subunits clustered with expansion=1 and inflation =3 identifies several INO80 subcomplexes. ( D) Proteins clustered like in (C) with crosslinks filtered by the identification score (cut-off 27). Workflow The ‘Protein Length’ file has to use the same protein names as the ‘Crosslink Data’ file and specifies the user- The typical workflow starts by selecting the input files and defined protein lengths as amino acid numbers. Similarly, the plot type in the ‘Input Parameters’ form. Optional fea- the ‘Annotations’ file links user-defined structural and func- tures of the schematic representations have to be activated tional annotations and their start and end positions with the at this step: (i) select automatic import of annotated sec- protein names. ondary structures from UniProt; (ii) select automatic im- The proteins in the diagrams are labeled with the port of annotated domains, families, active sides, binding names given in the ‘Crosslink Data’ file. We rec- sites, conserved sites, repeats and post-translational modi- ommend using the first part of the FASTA header fications from InterPro; (iii) to introduce user-defined pro- (db|UniqueIdentifier |EntryName) as protein descriptor tein lengths deselect the UniProt protein lengths in order which facilitates automatic import of protein lengths to access the respective upload mask; (iv) to group proteins and annotations through the primary accession number based on the crosslink density choose the clustering type; (UniqueIdentifier). The entry name or a user-defined name (v) to filter the crosslink dataset define the column heading replacing the entry name in the descriptor is displayed of the filter value; (vi) upload the Consurf ( 25) output files in the representations. The exclusive application of user- to display the evolutionary conservation of amino acids. defined names does not support the automatic import of The ‘Plot’ button generates the respective crosslink rep- protein lengths and annotation data and thus necessitates resentation in the same browser window. For each plot type the upload of the respective input files. the mouse over event on the crosslink line indicates the crosslink structure, the protein names, the absolute linkage positions and the selected score values. Mouse over on an- W366 Nucleic Acids Research, 2015, Vol. 43, Web Server issue Figure 3. Customized annotations in xVis.(A) Selective representation of the Rvb1/2 and Ino80/Ies2 modules with the subunit Arp8 and histone H2A in a network diagram showing annotations from InterPro (top bars in the protein representation) and user-defined domains (bottom bars). ( B) Evolutionary conservation of amino acid positions in the proteins Ino80 and Arp8 (bottom bars) obtained by the ConSurf webserver. (C) InterPro annotation legend used in (A) and (B). (D) User-defined domains displayed in (A). notation fields displays the respective protein information or hierarchically clustered (Figure 1C). Protein complexes with the sequence boundaries. are indicated by by the hierarchical arrangement which or- In the active plot the parameters of the plot type, sort ders the proteins according to the number of crosslinks. For type and filter value can be adjusted in the ‘Change Input this purpose we built a dendrogram by a bottom up algo- Parameters’ menu. The ‘Save and Restore Sessions’ menu rithm that starts at the proteins with the most inter-protein facilitates saving of all ‘Input Parameters’, ‘Shapes’, ‘Inter- crosslinks and uses the average number of restraints to cal- Pro Annotations’, ‘Filter’ and ‘Markov Clustering Param- culate the new cluster. Strikingly, hierarchical clustering, eters’ settings and of the protein positions in the network even in the absence of supporting structural and biochem- plot. A session starts with generating a plot in the ‘Input ical data, is sufficient to accurately assign INO80 subunits Parameters’ form. During the session you may switch plot to its subcomplexes (Figure 1A and C). types and change various parameters several times. Saving Several menus shown at the left-hand side of the dia- a session stores the parameters of the active plot and the gram facilitate the customization and interpretation of the protein positions of the network diagram in a file. Loading crosslink graph. The ‘Secondary Structures’ menu allows a session opens the latest diagram and restores all settings for selectively displaying the different secondary structures for further manipulation. and their colors. The ‘InterPro Annotations’ menu provides the possibility to choose the annotations which are rep- Circular and bar plots. The circular and bar plots (Fig- resented in the diagram. The quality of crosslink identifi- ure 1B–D) offer the same visualization features. The pro- cations is indicated by score values. The xQuest software teins are represented as arcs or bars which are scaled to calculates identification scores (Id-Score) and FDRs, how- the protein lengths and crosslinks are drawn as circular ever, also scores computed by different crosslink analysis lines. Protein secondary structures and annotations im- software can be selected in the ‘Input Parameters’ form ported from public databases are plotted into the protein as filter value. Using the slider in the ‘Filter’ menu selec- bars and arcs next to the sequence scale (Figure 1C and D) tively shows crosslinks below or above the chosen thresh- whereas user-defined domains are indicated in the opposite old. This menu further enables the selective display of inter- half of the objects (Figure 3A). and intra-protein crosslinks in different colors and marking For these plot types the crosslinked proteins are arranged individual proteins shows the selection of crosslinks that in- either alphabetically (Figure 1B), alphabetically in groups Nucleic Acids Research, 2015, Vol. 43, Web Server issue W367 Figure 4. Topology of a network of modular PP2A complexes displayed in a network diagram. Proteins were grouped by the MCL algorithm applying the parameters expansion=2 and inflation =2 (STRIPAK, B”’/striatin-interacting phosphatase and kinase, STRIPAK, complex; TCP1 ring complex, TRiC). tersect with these proteins. Once the layout of the circular INO80 subcomplexes (Figure 2C) and places the histones or bar diagram is optimized the graphs may be exported as H2B and H3 in a separate cluster. Similar to the hierarchi- SVG. cal clustering in the circular and bar plot, histone H2A is assigned to the Rvb1/2 module indicating spatial proximity within the INO80 - nucleosome complex. Network diagram. The network is generated by a force- directed layout. In contrast to the circular and bar plots, se- lecting proteins in the ‘Filter’ menu’ of the network diagram Evaluation and interpretation. A slider in the ‘Filter’ menu displays a selection of the subunits and their crosslinks. The of the individual plots facilitates the selective display of sorting type of the network diagram has to be specified in crosslinks according to identification scores (Figure 2D) or the ‘Input Parameters’ or ‘Change Input Parameters’ form FDRs. and similarly as for the circular plot, facilitates the grouping To further assess the quality of crosslink identifications of proteins that are bridged by crosslinks. The MCL Algo- xVis offers the possibility to evaluate the fragment ion spec- rithm (26) identifies clusters of proteins with high crosslink tra. Once the local installation of xVis is connected to a densities and separates them from low density areas. The xQuest server double-clicking on a crosslink line in a dia- ‘Markov Cluster Parameters’ menu helps to set the density gram opens the corresponding fragment ion spectrum in a restraints that define a cluster. The expansion and inflation separate tab. This allows the interactive manual inspection values determine the cluster boundaries whereas the thresh- of the spectral quality of individual crosslink identifications old excludes groups of proteins with numbers below this in the dataset. value from clustering. The analysis of the INO80 complex with xVis revealed Proteins of a cluster are indicated by the same color. In that clustering of proteins according to the crosslink den- the network diagram without sorting the 15 subunits of the sity identified subcomplexes that correspond to structural INO80 complex and the histones are same-colored (Fig- modules of the remodeler (Figure 2C). ure 2A). The MCL Algorithm applied with parameters for In addition to the subunit architecture, plotting anno- wider cluster boundaries identifies the Nhp10 module, how- tated protein domains and motifs visualizes their topology ever, cannot discriminate between the remaining INO80 and provides further structural and functional insights. The subunits and the histones (Figure 2B). Adjusting the pa- crosslinks of the H2A core structures to the Snf2 ATPase rameters to narrow cluster boundaries perfectly resolves all domain of INO80 and next to the AAA ATPases of Rvb1 W368 Nucleic Acids Research, 2015, Vol. 43, Web Server issue and Rvb2 indicate that the H2A/H2B dimer is in close prox- matic workflows providing 3D hybrid models of macro- imity to these ATPases and suggested an ATP-dependent molecular protein complexes at different levels of detail mechanical activity for histone exchange (Figure 3A, C and (17,28). The schematic representations by xVis and other D) (21). web servers visualize the subunit topology by position- As amino acids of functionally important sites, like bind- ing protein bars or nodes according to intermolecular ing interfaces or catalytic sites, tend to be evolutionary con- crosslinks. Replacing these protein objects in future imple- served, xVis facilitates the import of evolutionary conser- mentations by simplified subunit shapes derived from in- vation rates estimated by the Consurf web server (25). The tramolecular restraints may delineate the 2D topology of Consurf conservation scores are plotted into the protein ob- protein complexes similar to 3D coarse-grained representa- jects following the scale from variable (1) to conserved (9) tions. amino acids (Figure 3B). Remarkably, the circular and network diagrams obtained by xVis indicate the spatial proximity of annotated protein regions. The structural information at the level of protein DISCUSSION domains or motifs facilitates the prediction of structural Here, we present the web server xVis for the fast and inter- and functional models and the design of experiments to test their relevance in vitro and in vivo. active visualization and interpretation of spatial restraints identified by different mass spectrometric workflows. The demand for tools for the 2D visualization of crosslinks is un- SUPPLEMENTARY DATA derscored by the software xiNet which was published during Supplementary Data are available at NAR Online. preparation of this manuscript (27)(http://crosslinkviewer. org/). xiNet represents crosslink data as node-link diagrams at residue resolution using an elaborate web-design for the FUNDING manipulation of the graphs and for the visualization of an- Junior researcher grant of the LMUexcellent initia- notated protein regions. tive; Bavarian Research Center of Molecular Biosystems; xVis offers, in addition to the network diagram, a circu- Deutsche Forschungsgemeinschaft [GRK1721]. Funding lar or bar plot which in many cases is the preferred rep- for open access charge: LMUexcellent initiative. resentation of spatial restraints on oligomeric complexes. Conflict of interest statement. None declared. 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xVis: a web server for the schematic visualization and interpretation of crosslink-derived spatial restraints

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Oxford University Press
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The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
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0305-1048
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1362-4962
DOI
10.1093/nar/gkv463
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25956653
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W362–W369 Nucleic Acids Research, 2015, Vol. 43, Web Server issue Published online 08 May 2015 doi: 10.1093/nar/gkv463 xVis: a web server for the schematic visualization and interpretation of crosslink-derived spatial restraints 1 1 2 1,* Maximilian Grimm , Tomasz Zimniak , Abdullah Kahraman and Franz Herzog Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universitat ¨ Munchen, ¨ Munich 81377, Germany and Institute of Molecular Life Sciences, University of Zurich, Zurich 8057, Switzerland Received February 25, 2015; Revised April 17, 2015; Accepted April 27, 2015 ABSTRACT only label surface exposed residues but also covalently link residues and thus reveal their proximity within the three- The identification of crosslinks by mass spectrom- dimensional (3D) structure of biomolecules and their com- etry has recently been established as an integral plexes. part of the hybrid structural analysis of protein com- In recent years, advances in mass spectrometric instru- plexes and networks. The crosslinking analysis de- mentation that facilitate the detection of modified pep- termines distance restraints between two covalently tides at high mass accuracy, resolution and sensitivity re- linked amino acids which are typically summarized sulted in the development of different workflows for the identification of crosslinked peptides in complex protein in a table format that precludes the immediate and samples (2,3). Chemical crosslinking combined with mass comprehensive interpretation of the topological data. spectrometry predominantly involves reagents carrying N- xVis displays crosslinks in clear schematic represen- hydroxy-succinimidyl esters which are reactive against pri- tations in form of a circular, bar or network diagram. mary amines of lysine side chains and protein N-termini. The interactive graphs indicate the linkage sites and Chemistries targeting acidic amino acids have been success- identification scores, depict the spatial proximity of fully introduced (4), however, have not yet led to routine ap- structurally and functionally annotated protein re- plications. Similar spatial information is obtained by com- gions and the evolutionary conservation of amino bining mass spectrometry with the site-specific incorpora- acids and facilitate clustering of proteins into sub- tion of photoactivatable amino acids which give rise to rad- complexes according to the crosslink density. Fur- icals highly reactive against various side chains (5). The reported protocols for the identification of thermore, xVis offers two options for the qualitative crosslinked residues differ by the physicochemical prop- assessment of the crosslink identifications by filter- erties of the spacer arm (6) and by the dedicated analysis ing crosslinks according to identification scores or software. In particular, recent workflows implemented false discovery rates and by displaying the corre- isotope-coded and collision-induced-dissociation (CID) sponding fragment ion spectrum of each crosslink cleavable spacer arms. Isotope-tagging introduces an for the manual validation of the mass spectrometric isotopic mass shift which detects crosslinks as isotopic data. Our web server provides an easy-to-use tool pairs and aids in identifying the fragment ions derived from for the fast topological and functional interpretation the two crosslinked peptides by the search engine xQuest of distance information on protein complex architec- (7). CID cleavable crosslinkers generate reporter ions and tures and for the evaluation of crosslink fragment give rise to the linear peptides modified with the remaining ion spectra. xVis is available under a Creative Com- crosslinker masses which facilitates the identification of the mons Attribution-ShareAlike 4.0 International license crosslink sites (8). Regardless of the workflow, the structural informa- at http://xvis.genzentrum.lmu.de/. tion determined by chemical and photoactivatable (9–11) crosslinking is a distance restraint which is based on the INTRODUCTION length of the reagent. We are using disuccinimidyl suberate as a crosslinker which has a spacer arm length of 11.4 A. Chemical labeling of functional groups has a long-standing In our study on a network of modular protein phosphatase history in the structural analysis of proteins and ribonucleic 2A (PP2A) complexes, crosslinks were evaluated by measur- acids (1). Initially, the tagging with different chemistries ing the distances between the two bridged lysines on avail- such as hydrogen/deuterium exchange or hydroxyl radical able X-ray structures and homology models (12). The me- labeling detected solvent accessible residues, also known dian Euclidian C-C distances of 287 intra-protein and as footprinting. In contrast, bifunctional reagents do not To whom correspondence should be addressed. Tel: +49 89 2180 76755; Fax: +49 89 2180 76999; Email: [email protected] C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2015, Vol. 43, Web Server issue W363 70 inter-protein crosslinks were 15.4 and 19.6 A, respec- representation below or above a certain cut-off value. For tively. The recently released Xlink Analyzer software enables crosslink datasets identified by the search engine xQuest, the automatic analysis and visualization of crosslinks in the we offer the possibility to connect to axQuest server for context of the 3D structures (13). In comparison to Euclid- the manual inspection of the crosslink fragment ion spec- ian distances the simulation of distance restraints on the tra. We also implemented sorting algorithms that arrange solvent accessible protein surface has been shown to be su- and group subunits according to crosslink densities. Strik- perior in constraining computational molecular modeling ingly, all subunits of the INO80 - nucleosome complex are and docking experiments (14,15). Inter-protein crosslinks correctly assigned to the previously identified subcomplexes indicate direct protein–protein contacts and will thus sig- solely based on hierarchical clustering or the Markov Clus- nificantly support systems biology efforts in predicting and ter (MCL) Algorithm using crosslink-derived restraints. reconstructing protein interactions of biological networks (16). MATERIALS AND METHODS The potential of crosslinks in integrating structural data xVis setup of different sources has been demonstrated by several groups. In particular, the heterogeneity of the sample prepa- xVis is a server-client-based software solution which uses rations and the intrinsic flexibility of macromolecular pro- PHP to fetch protein information from the InterPro (http:// tein complexes have in many cases prevented their structural www.ebi.ac.uk/interpro/) and UniProt (http://www.uniprot. elucidation by established high-resolution methods. These org/) databases. The protein lengths and secondary struc- limitations led to the design of hybrid approaches that ap- tures from UniProt are accessed with dbfetch (22) provided ply crosslink-derived distance restraints and computational by EMBL-EBI using UniProt identifiers. The annotations modeling to integrate crystal structures and models of in- are downloaded from InterPro with MartWizard (23). The dividual subunits with electron microscopic (EM) densities diagrams are generated on the client by a JavaScript library and subunit localization (17–19). Depending on the input named D3 (Data-Driven Documents) (24). D3 is used to data the integrative structural analysis results in models at generate scalable vector graphics (SVG) and to animate the different levels of detail. In recent studies we have obtained arrangements of proteins in the network diagram with force a hybrid model of RNA polymerase I at near atomic res- layout. To get a similar design on each browser we used olution (20) whereas the availability of only sparse high- Bootstrap and Jasny Bootstrap. resolution structural data on the INO80 complex resulted xVis can be installed on a server with PHP by coping the in a topological model of less detail (21). source code into the web directory of the server. We also The budding yeast INO80 complex is composed of provide a portable version containing the XAMPP server 15 subunits and belongs to the INO80/SWR1 family for Windows. The local installation of xVis is important for chromatin remodelers. Its important role in transcrip- its connection to the xQuest server. The URL of the xQuest tion and genome maintenance is the exchange of histone server has to be entered on the ‘Settings’ page. H2A.Z/H2B dimers with free H2A/H2B. To elucidate the mechanism of histone exchange we analyzed the architec- Input data ture of the INO80 complex interacting with its nucleosome substrate by combining EM 3D densities and subunit lo- The input data for the visualization of crosslink datasets calization with chemical crosslinking and mass spectrome- comprises the coordinates of crosslinks including protein try. This hybrid structural analysis revealed distinct struc- descriptors and positions of the linked residues, protein tural modules of an embryo-shaped complex that embraces lengths information, quality scores for the mass spectro- the nucleosome like a flexible clamp. Chemical crosslinking metric identification, annotation of protein domains and together with subunit localization and biochemical assays xQuest references which provide the possibility to link each identified individual subcomplexes and assigned them to crosslink with the corresponding fragment ion spectrum. structural models (Figure 1A). Furthermore, the crosslink- The input data are uploaded as comma separated files in derived spatial restraints were used to draw a proximity the ‘Input Parameters’ form. In addition to the main input map of structurally and functionally annotated domains file, two accessory files help to customize the representa- which provided detailed insights into the mode of nucleo- tions. xVis identifies the input values by the column head- some binding and histone H2A variant exchange. ings, hence, the order of columns is not important whereas We thus developed xVis, a software solution for the fast the headings have to precisely match the names given in the and schematic visualization and interpretation of distance sample input files which can be retrieved from the ‘Input information on protein complexes and networks. xVis is Parameters’ and ‘Downloads’ page. able to display crosslink-derived distances obtained by dif- The main ‘Crosslink Data’ file is derived from the xQuest ferent workflows in form of a circular, bar or network plot. output file, however, several crosslink datasets obtained by The import of structurally and functionally annotated do- other workflows can be visualized if delivered in the appro- mains as well as the evolutionary conservation rates of priate input format. The ‘Crosslink Data’ file has to provide amino acids is crucial for the initial structural and func- the protein names and the position of the linkage sites and tional interpretation prior to the more laborious genera- optionally, may contain the identification score, the FDR tion of 3D models. Furthermore, the program offers two and the xQuest reference of the fragment ion spectra. The options for the evaluation of crosslink identifications. The xQuest reference of each crosslink is generated from val- filtering of crosslinks according to the identification score ues of different columns, hence requiring a complete xQuest or the false discovery rate (FDR) allows their selective output file as ‘Crosslink Data’ input file. W364 Nucleic Acids Research, 2015, Vol. 43, Web Server issue Figure 1. Topological analysis of the INO80 chromatin remodeler in complex with its nucleosome substrate. (A) Identification of subcomplexes and as- signment to different structural modules using EM, chemical crosslinking and biochemical assays. (B) Crosslinked INO80 subunits are visualized and alphabetically sorted in a circular representation using xVis. The protein names are colored according to the modules in (A). (C) Hierarchical clustering of INO80 subunits based on crosslinks displayed in a circular diagram by xVis.(D) Hierarchical clustering of INO80 subunits based on crosslinks shown in a bar diagram by xVis. Bars represent InterPro domains (Figure 3A) (inter-protein crosslinks in black, intra-protein crosslinks in red). Nucleic Acids Research, 2015, Vol. 43, Web Server issue W365 Figure 2. Topological analysis of the INO80 - nucleosome complex by applying the Markov Cluster (MCL) Algorithm in the network diagram. (A) Subunits non-clustered (all proteins blue colored). (B) Subunits clustered with the MCL parameters expansion=2 and inflation =1 separates the Nhp10 subcomplex (light blue). (C) Subunits clustered with expansion=1 and inflation =3 identifies several INO80 subcomplexes. ( D) Proteins clustered like in (C) with crosslinks filtered by the identification score (cut-off 27). Workflow The ‘Protein Length’ file has to use the same protein names as the ‘Crosslink Data’ file and specifies the user- The typical workflow starts by selecting the input files and defined protein lengths as amino acid numbers. Similarly, the plot type in the ‘Input Parameters’ form. Optional fea- the ‘Annotations’ file links user-defined structural and func- tures of the schematic representations have to be activated tional annotations and their start and end positions with the at this step: (i) select automatic import of annotated sec- protein names. ondary structures from UniProt; (ii) select automatic im- The proteins in the diagrams are labeled with the port of annotated domains, families, active sides, binding names given in the ‘Crosslink Data’ file. We rec- sites, conserved sites, repeats and post-translational modi- ommend using the first part of the FASTA header fications from InterPro; (iii) to introduce user-defined pro- (db|UniqueIdentifier |EntryName) as protein descriptor tein lengths deselect the UniProt protein lengths in order which facilitates automatic import of protein lengths to access the respective upload mask; (iv) to group proteins and annotations through the primary accession number based on the crosslink density choose the clustering type; (UniqueIdentifier). The entry name or a user-defined name (v) to filter the crosslink dataset define the column heading replacing the entry name in the descriptor is displayed of the filter value; (vi) upload the Consurf ( 25) output files in the representations. The exclusive application of user- to display the evolutionary conservation of amino acids. defined names does not support the automatic import of The ‘Plot’ button generates the respective crosslink rep- protein lengths and annotation data and thus necessitates resentation in the same browser window. For each plot type the upload of the respective input files. the mouse over event on the crosslink line indicates the crosslink structure, the protein names, the absolute linkage positions and the selected score values. Mouse over on an- W366 Nucleic Acids Research, 2015, Vol. 43, Web Server issue Figure 3. Customized annotations in xVis.(A) Selective representation of the Rvb1/2 and Ino80/Ies2 modules with the subunit Arp8 and histone H2A in a network diagram showing annotations from InterPro (top bars in the protein representation) and user-defined domains (bottom bars). ( B) Evolutionary conservation of amino acid positions in the proteins Ino80 and Arp8 (bottom bars) obtained by the ConSurf webserver. (C) InterPro annotation legend used in (A) and (B). (D) User-defined domains displayed in (A). notation fields displays the respective protein information or hierarchically clustered (Figure 1C). Protein complexes with the sequence boundaries. are indicated by by the hierarchical arrangement which or- In the active plot the parameters of the plot type, sort ders the proteins according to the number of crosslinks. For type and filter value can be adjusted in the ‘Change Input this purpose we built a dendrogram by a bottom up algo- Parameters’ menu. The ‘Save and Restore Sessions’ menu rithm that starts at the proteins with the most inter-protein facilitates saving of all ‘Input Parameters’, ‘Shapes’, ‘Inter- crosslinks and uses the average number of restraints to cal- Pro Annotations’, ‘Filter’ and ‘Markov Clustering Param- culate the new cluster. Strikingly, hierarchical clustering, eters’ settings and of the protein positions in the network even in the absence of supporting structural and biochem- plot. A session starts with generating a plot in the ‘Input ical data, is sufficient to accurately assign INO80 subunits Parameters’ form. During the session you may switch plot to its subcomplexes (Figure 1A and C). types and change various parameters several times. Saving Several menus shown at the left-hand side of the dia- a session stores the parameters of the active plot and the gram facilitate the customization and interpretation of the protein positions of the network diagram in a file. Loading crosslink graph. The ‘Secondary Structures’ menu allows a session opens the latest diagram and restores all settings for selectively displaying the different secondary structures for further manipulation. and their colors. The ‘InterPro Annotations’ menu provides the possibility to choose the annotations which are rep- Circular and bar plots. The circular and bar plots (Fig- resented in the diagram. The quality of crosslink identifi- ure 1B–D) offer the same visualization features. The pro- cations is indicated by score values. The xQuest software teins are represented as arcs or bars which are scaled to calculates identification scores (Id-Score) and FDRs, how- the protein lengths and crosslinks are drawn as circular ever, also scores computed by different crosslink analysis lines. Protein secondary structures and annotations im- software can be selected in the ‘Input Parameters’ form ported from public databases are plotted into the protein as filter value. Using the slider in the ‘Filter’ menu selec- bars and arcs next to the sequence scale (Figure 1C and D) tively shows crosslinks below or above the chosen thresh- whereas user-defined domains are indicated in the opposite old. This menu further enables the selective display of inter- half of the objects (Figure 3A). and intra-protein crosslinks in different colors and marking For these plot types the crosslinked proteins are arranged individual proteins shows the selection of crosslinks that in- either alphabetically (Figure 1B), alphabetically in groups Nucleic Acids Research, 2015, Vol. 43, Web Server issue W367 Figure 4. Topology of a network of modular PP2A complexes displayed in a network diagram. Proteins were grouped by the MCL algorithm applying the parameters expansion=2 and inflation =2 (STRIPAK, B”’/striatin-interacting phosphatase and kinase, STRIPAK, complex; TCP1 ring complex, TRiC). tersect with these proteins. Once the layout of the circular INO80 subcomplexes (Figure 2C) and places the histones or bar diagram is optimized the graphs may be exported as H2B and H3 in a separate cluster. Similar to the hierarchi- SVG. cal clustering in the circular and bar plot, histone H2A is assigned to the Rvb1/2 module indicating spatial proximity within the INO80 - nucleosome complex. Network diagram. The network is generated by a force- directed layout. In contrast to the circular and bar plots, se- lecting proteins in the ‘Filter’ menu’ of the network diagram Evaluation and interpretation. A slider in the ‘Filter’ menu displays a selection of the subunits and their crosslinks. The of the individual plots facilitates the selective display of sorting type of the network diagram has to be specified in crosslinks according to identification scores (Figure 2D) or the ‘Input Parameters’ or ‘Change Input Parameters’ form FDRs. and similarly as for the circular plot, facilitates the grouping To further assess the quality of crosslink identifications of proteins that are bridged by crosslinks. The MCL Algo- xVis offers the possibility to evaluate the fragment ion spec- rithm (26) identifies clusters of proteins with high crosslink tra. Once the local installation of xVis is connected to a densities and separates them from low density areas. The xQuest server double-clicking on a crosslink line in a dia- ‘Markov Cluster Parameters’ menu helps to set the density gram opens the corresponding fragment ion spectrum in a restraints that define a cluster. The expansion and inflation separate tab. This allows the interactive manual inspection values determine the cluster boundaries whereas the thresh- of the spectral quality of individual crosslink identifications old excludes groups of proteins with numbers below this in the dataset. value from clustering. The analysis of the INO80 complex with xVis revealed Proteins of a cluster are indicated by the same color. In that clustering of proteins according to the crosslink den- the network diagram without sorting the 15 subunits of the sity identified subcomplexes that correspond to structural INO80 complex and the histones are same-colored (Fig- modules of the remodeler (Figure 2C). ure 2A). The MCL Algorithm applied with parameters for In addition to the subunit architecture, plotting anno- wider cluster boundaries identifies the Nhp10 module, how- tated protein domains and motifs visualizes their topology ever, cannot discriminate between the remaining INO80 and provides further structural and functional insights. The subunits and the histones (Figure 2B). Adjusting the pa- crosslinks of the H2A core structures to the Snf2 ATPase rameters to narrow cluster boundaries perfectly resolves all domain of INO80 and next to the AAA ATPases of Rvb1 W368 Nucleic Acids Research, 2015, Vol. 43, Web Server issue and Rvb2 indicate that the H2A/H2B dimer is in close prox- matic workflows providing 3D hybrid models of macro- imity to these ATPases and suggested an ATP-dependent molecular protein complexes at different levels of detail mechanical activity for histone exchange (Figure 3A, C and (17,28). The schematic representations by xVis and other D) (21). web servers visualize the subunit topology by position- As amino acids of functionally important sites, like bind- ing protein bars or nodes according to intermolecular ing interfaces or catalytic sites, tend to be evolutionary con- crosslinks. Replacing these protein objects in future imple- served, xVis facilitates the import of evolutionary conser- mentations by simplified subunit shapes derived from in- vation rates estimated by the Consurf web server (25). The tramolecular restraints may delineate the 2D topology of Consurf conservation scores are plotted into the protein ob- protein complexes similar to 3D coarse-grained representa- jects following the scale from variable (1) to conserved (9) tions. amino acids (Figure 3B). Remarkably, the circular and network diagrams obtained by xVis indicate the spatial proximity of annotated protein regions. The structural information at the level of protein DISCUSSION domains or motifs facilitates the prediction of structural Here, we present the web server xVis for the fast and inter- and functional models and the design of experiments to test their relevance in vitro and in vivo. active visualization and interpretation of spatial restraints identified by different mass spectrometric workflows. The demand for tools for the 2D visualization of crosslinks is un- SUPPLEMENTARY DATA derscored by the software xiNet which was published during Supplementary Data are available at NAR Online. preparation of this manuscript (27)(http://crosslinkviewer. org/). xiNet represents crosslink data as node-link diagrams at residue resolution using an elaborate web-design for the FUNDING manipulation of the graphs and for the visualization of an- Junior researcher grant of the LMUexcellent initia- notated protein regions. tive; Bavarian Research Center of Molecular Biosystems; xVis offers, in addition to the network diagram, a circu- Deutsche Forschungsgemeinschaft [GRK1721]. Funding lar or bar plot which in many cases is the preferred rep- for open access charge: LMUexcellent initiative. resentation of spatial restraints on oligomeric complexes. Conflict of interest statement. None declared. 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Published: Jul 1, 2015

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