Tutorial: Using the SFLD and Cytoscape to Make Hypotheses About Enzyme Function for an Isoprenoid Synthase Superfamily Sequence

Transcription

1 Tutorial: Using the SFLD and Cytoscape to Make Hypotheses About Enzyme Function for an Isoprenoid Synthase Superfamily Sequence Requirements: 1. A web browser 2. The cytoscape program (available for download from: Note: Cytoscape version 2 is used in this tutorial. The capabilities shown are available in version 3, but the process to access them may differ slightly. This tutorial will take you on a brief tour of the Structure Function Linkage Database and the cytoscape network visualization software, which we use to visualize the sequence relationships between large groups of related enzymes, like those in the various Isoprenoid Synthase superfamilies. We'll take a sequence that hasn t been experimentally characterized and use the SFLD and cytoscape to make some hypotheses about its function. Go to: 1

2 We'll start with a brief introduction to the SFLD. The purpose of the SFLD is to link enzyme sequence and structural information to specific functional information. There are a couple of ways to get into the database. We'll start by browsing through the available superfamilies. Click the Browse by Superfamily link at the top of the page The core superfamilies are those that are most highly curated. There are a larger number of superfamilies in the extended SFLD, but the curation is less deep. Sequences in each SFLD superfamily are organized hierarchically, based on sequence, structure and function. At the top level of the hierarchy is an enzyme superfamily. Enzymes in the same superfamily are evolutionarily related, and although any given pair of enzymes within the superfamily may have diverged quite a bit and share low levels of overall sequence identity, superfamily enzymes all share conserved active site residues that mediate a common chemical capability. There are a few superfamilies in the SFLD that contain enzymes that perform isoprenoid synthase reactions. These are: Aromatic Prenyltransferase, Isoprenoid Synthase Type I, Isoprenoid Synthase Type II, and Di-trans-poly-cis-decaprenylcistransferase. I'll be using the Isoprenoid Synthase Type I superfamily in this demo. 2

3 After superfamily, the next level of the hierarchy is subgroup. In the Isoprenoid synthase I SF, for example, there are 14 different subgroups. These subgroups have been defined based on overall sequence similarity, so that enzyme in each subgroup are more similar to each other than they are to other enzymes in the superfamily. After subgroup, the next level down in the hierarchy is family. Enzymes in a family have more sequence similarity to each other than to other sequences within the parent subgroup, but more importantly, they're thought to catalyze the same overall reaction based on the same mechanism. This does get a little complicated for some of the enzymes in the isoprenoid synthase superfamilies, because some are highly promiscuous. For this reason, when an enzyme is known to have multiple products, we only put that enzyme into a family if it catalyzes the formation of one particular product with at least 50% yield. We currently have 63 families defined for IS I. Click the Isoprenoid Synthase Type I link. At the top of the Isoprenoid Synthase Type I superfamily page you'll see some basic summary information, for example, the total number of nonredundant sequences in the superfamily. These are subdivided into those with a family assignment (known function) and those without a family assignment (unknown function). You can also see the total number of structures and reactions, and an image of an example superfamily structure. Under the Description tab, you'll see a brief summary of the reaction chemistry that is typical of the superfamily. The References tab gives a few general literature references for further reading. In the next section, you can access the curated alignment for the group and also some download tools. We'll explore those later. 3

4 The bottom section of the page shows a tabular view of the way the sequences and structures are distributed between subgroups and families. Subgroups are shown with a blue background and families with a yellow background. Note that, in some cases, there is more than one level of subgroup. For example, the terpene cyclase like 1 C-term subgroup has some additional subgroups defined within it: TPS-g, TPS-b, TPS-d, TPS-a, TPS-c, TPS-e, and TPS-f these are more closely related groups of enzymes than across the subgroup as a whole, but enzymes within them may still catalyze different overall reactions. For example, the TPS-g subgroup contains three different families (myrcene synthase, (3S,6E)-nerolidol synthase, (E)-beta-ocimene synthase) corresponding to three different overall reactions. At the top of the table, the column with the T heading lists the total number of sequences in the group. The K column lists the number of sequences in the group with family assignments (those with known function), the U column lists the number of sequences in 4

5 the group without family assignments (those with unknown function) and the S column lists the number of structures in the group. All of the subgroup names, family names, reaction names, sequence and structure counts are clickable links, so if you want more detail about anything, you can click on the associated link. Click on the sequence link for the taxadiene synthase family (red arrow in above image). In the sequence table that appears, click on any of the links in the Name column. Many of the sequence in the Isoprenoid synthase type I superfamily have a multidomain architecture where a given enzyme may have one domain in the IS I superfamily and 5

6 another in a different superfamily. You'll be able to identify possible multidomain proteins by looking at the sequence section of the FunctionalDomain page (above). The domain thought to be part of the Isoprenoid Synthase Type I superfamily is shown in black, while the remaining portion of the sequence is in gray. Sequences with a large region in gray may be multidomain proteins. If the other domain is a member of an SFLD superfamily, you'll be able to access it via the link in the Related Enzyme Functional Domain(s) section. That was a brief introduction to the SFLD. Now we're going to explore the database in more detail. We'll start with an enzyme that has no associated experimental information, and use the SFLD and cytoscape to make some hypotheses about its function. In another browser tab or window, go to: This is the NCBI GenPept page for an enzyme annotated as a squalene synthase. However, it doesn't have any associated literature references, so the annotation doesn t seem to be based on experimental data. 6

7 Click on the FASTA link (indicated by the red arrow in the above image) and copy the protein sequence. Back in the SFLD tab or window, click on the Search by Enzyme link at the top of the page. There are two different ways that you can search the SFLD using a protein sequence. We'll start by doing a BLAST search against all the SFLD sequences. Paste the sequence into the Protein Sequence box, make sure the BLAST radio button is filled in, and click the Search button. At the top of the results page, you'll see a summary of the BLAST hits. This summary table lists the superfamilies, subgroups, and families assigned to the individual sequences found as BLAST hits. Note that they are ordered based on the SFLD hierarchy rather than BLAST E-value. For each superfamily, subgroup, or family, you'll see the number of BLAST hits from the group. You'll also see the maximum and minimum E-values and scores of hits from that group, so you can get an idea of how well your query matches the group. If you want to explore how well your sequence matches the group in more detail, you can look at how well your sequence aligns to the group using the links in the ShowAlignment column. In some cases, you can also view your query sequence in the context of a cytoscape sequence similarity network of the group, via the buttons in the Show Network column. We'll explore sequence alignments and cytoscape networks later. 7

8 Underneath the summary section, you'll see a list of the top 250 individual BLAST hits, with the best matches listed first. The name of each hit as well as some associated annotation information is listed. You'll also see the E-value and score of the match. If you want to explore the match in more detail you can click on the Show button in the Alignment column to view the BLAST alignment of your query sequence with that hit. Let's go back to the summary section and look at the results in a little more detail. As we would expect, all the hits were from the same SFLD superfamily the Isoprenoid Synthase Type I superfamily. The top subgroup match was the squalene/phytoene synthase subgroup, which is consistent with the NCBI annotation of squalene synthase. But the top family hit comes from the dehydrosqualene synthase family. In fact, the squalene synthase family isn't even present in the top 250 BLAST hits. So, just based on BLAST scores, it looks like the sequence might actually be a dehydrosqualene synthase. But lets look in a little more detail at how well the sequence fits in to both families. Click the back button on your browser to return to the Search By Enzyme page. Click the HMM radio button and then click the Search button Now, instead of doing a BLAST search, we're searching against the SFLD hidden Markov models these are models based on sequence alignments, which give the probability of finding a particular residue in a particular column of the multiple alignment. The SFLD HMMs are based on curated multiple sequence alignments for all the families, subgroups, and superfamilies in the database. 8

9 The results list the groups that the sequence matches. Note that these matches are sorted according to the SFLD hierarchy rather than E-value. For each match, you'll see the name of the group, it's level within the SFLD hierarchy, and the E-value and score of the match. So you can see here that our sequence does match the squalene synthase family, though the E-value for the match with the dehydrosqualene synthase family is more significant. But E-value can only give us a general idea of how well the sequence matches to these families, so let's look at the matches in more detail. Click the "Align to this Family" link for the squalene synthase family. You'll see a curated alignment for the squalene synthase family, with the sequence of interest added at the bottom. The catalytic residues required for family-specific functionality are listed in the table at the bottom of the page. Each residue is associated with an evidence code that tells you what kind of information was used to determine its functional role. Click the icon next to one of the Evidence Codes. 9

10 Clicking on the link next to an evidence code will bring up a tool tip that explains what it means. Click on a reference link. 10

11 When applicable, residues are also associated with a literature reference. Clicking on the reference link brings up the abstract of the relevant paper. The catalytic residues are also highlighted in the alignment, so it s easy to scroll through and see that, although the sequence of interest does have most of the catalytic residues, it's missing the final phenylalanine required for the second part of the reaction. So it looks like the Genbank annotation of squalene synthase may be wrong. Let's see how this sequence fits with the dehydrosqualene synthase family. Click the back button on your browser to return to the HMM search results. (You may have to click another button to re-request the document from the website.) Click on the "Align to this Family" link for the dehydrosqualene synthase family. You'll see the curated alignment for the dehydrosqualene synthase family. Again, the sequence of interest has been added at the bottom. Scrolling through, you can see that, in addition to good overall sequence similarity, the sequence has all the catalytic residues known to be required for family-specific functionality. It looks like the sequence may be a dehydrosqualene synthase, so let's get more information about the family. Click the Dehydrosqualene synthase link at the top of the page (indicated by the red arrow in the above image). You'll see the family page for the dehydrosqualene synthase family. The top of the page is organized in a similar manner to the superfamily page you already saw. 11

12 Further down is an active site image, with the important catalytic residues shown. Clicking on this image will open the associated structure in the molecular graphics program Chimera (Chimera must be installed on your computer. Free download for academic use: so that you can explore it interactively. Further down is the overall reaction catalyzed by the family. Scroll back to the top of the page and click the Sequences link. 12

13 You'll see a table listing all the sequences in the family as well as some associated annotation information, like species and links to other databases. Click the "Toggle Columns" tab. You can choose what information is shown in the table via the buttons available at the top of page. You can also download the information as a Tab delimited file, which you can open in any spreadsheet program like Excel, by clicking on the TSV button at the top right of the page. You can download the sequence set as a fasta file by clicking the "FULL Fasta" button at the top right of the page. Based on our SFLD tools, it looked like our sequence is a dehydrosqualene synthase, but it might be useful to get a broader picture of how the sequence relates to the other enzymes in the squalene/phytoene synthase like subgroup. We can do that using cytoscape, a software program that lets you visualize sequence similarity relationships as 13

14 networks. In this next part of the tutorial, you'll learn how to explore these networks interactively in order to develop hypotheses about structure-function relationships in enzyme superfamilies. Click on the "Squalene/Phytoene Synthase Like" link at the top of the page. The information on this subgroup page is organized similarly to the superfamily and family pages you've already seen. In the middle section, click on the Download Network tab. In this section, you can access two different types of networks a full network, where all the sequences in the subgroup will be represented as unique nodes in the network, or a representative network, where similar sequences have been collapsed into the same node. The 50% ID listed on the button for the representative network means that the nodes have been created by clustering the subgroup sequence into 50% ID clusters using the CD-HIT 14

15 program. Each node in the representative network represents the sequences in one of those CD-HIT clusters. The networks in the SFLD are created using BLAST as a sequence similarity metric, so you can choose the BLAST e-value cutoff when you download a full network. The ideal value will depend on the group you re looking at and the questions you re asking. Type "1e-50" in the box next to "Download a Full Network Based on E-value Cutoff", and click the Full network button. When prompted, save the file. Decompress the resulting zip file. You'll see several different files. fullnetdownloads.readme.txt is a text file that explains what information is contained in each of these files, and also gives you some literature references about sequence similarity networks. Open the cytoscape program and choose File - Import - Network (Multiple File Types) from the top Menu bar. 15

16 In the Import Network box, click the Select button. In the Import Network Files browser, navigate to network_subgroup_1018.xgmml, click the file name, and click the Open button. Back in the Import Network box, click the Import button. After the network has loaded, click the Close button in the Loading Network box. NOTE: If you're having trouble opening larger networks, you may need to increase the memory for Cytoscape. See: When the network first opens up, you'll have a very uninformative view. So the first thing you'll want to do is lay the network out. There are many layouts available with Cytoscape. In addition to the organic layout that we'll use in a minute, you might also want to try some of the edge-weighted layouts. In the top menu bar, choose Layout - yfiles - Organic. 16

17 Each of these squares, or nodes, represents a single sequence in the squalene/phytoene synthase like subgroup, and each of the lines, or edges, represents a BLAST connection at least as significant as 1* In organic layout, the edges between sequences only represent connectivity that is, they show that two nodes are connected at the specified e-value cut-off. But while the lengths of these edges don't directly represent how distant these proteins are from each other, they give a pretty good approximation. For more information, see the Atkinson reference in the fullnetdownloads.readme.txt file, available with your network download. Sequences are colored by family (there are relatively few sequences in this subgroup with a family assignment, and thus most sequences in the network are not colored), and sequences in the same family cluster together. Click and drag your mouse to highlight some of the nodes in the cluster at the top middle of the screen that includes some brown nodes. 17

18 Clicking on a node selects that sequence, and you ll see it highlighted in yellow on the network. Sequences are also associated with annotation information, which is pulled over from the SFLD. (A list of all the available attributes and their definitions is given in the fullnetattributes.txt file, available as part of the network download.) You can see annotation information for the sequence or sequences you ve selected in the Data Panel at the bottom of the screen. Click the top left button in the Data Panel (indicated by the red arrow in the picture above) to select the annotation information you wish to view. (For example: Domain length, Family, Family evidence code, PDB IDs, Species, Type of life, and full length). 18

19 The Family column lists the SFLD family classification, and, related to that, the Family evidence code column lists the type of evidence used to assign the sequence to the family. The IEA evidence code, which stands for Inferred from Electronic Annotation, is given to sequences that are assigned to a family by automated scripts based on their sequence similarity, so they're the least reliable classifications. Sequences with the CFM (Canonical Family Member) evidence code have been experimentally determined to catalyze the family specific reaction, so those are the most reliable classifications. A list of all the evidence codes and their definitions is available on the SFLD website ( There are two columns for length--domain length gives the length of that portion of the sequence thought to be part of the isoprenoid synthase type I superfamily, and full length gives the length of the full sequence. The PDB IDs column lists the Protein Databank IDs for sequences with at least 95% ID to a solved crystal structure. You'll also see species information, and a more general category that we call type of life, which can be useful if you're interested in enzymes from a particular taxonomic classification. You can sort by any column by clicking on the name. One of the great features of cytoscape is that you can color the network according to any of the annotation information. For example, instead of coloring this network according to SFLD family, it could be colored according to the type of life classification of each node. In the left panel of the display, click the arrow to the right of Network to go to the Vizmapper Click on the button indicated with the red arrow and select Create new Visual Style from the popup menu 19

20 In the Enter Visual Style Name box, enter the name for your new style (ex. Type of Life) and click the OK button 20

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22 The Visual Mapping Browser section shows a list of all the properties of the network that can be changed. We will only be changing Node Color, but a similar process can be used to change any of the other properties. Double click Node Color (indicated by the red arrow in the above image) to send it to the top of the list. Click on "Please select a mapping ", then click on "Discrete Mapper" in the popup menu. Click on "Please select a value!", then click on "Type of Life" in the popup menu. Click on "Node Color", then right click on it and select Generate Discrete Values Rainbow 1 from the popup menu This tells cytoscape to automatically choose a color for each unique type of life. 22

23 You can change any color you don't like manually by clicking on the color, clicking on the box with three dots, and then choosing a new color from the set of swatches and clicking the OK button. 23

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25 Notice that there are some white nodes that are hard to see because they blend into the background. These are nodes with no value for the type of life attribute. Their color can be changed by changing the default node color. Click in the Defaults box. In the popup menu, click on NODE_FILL_COLOR. In the Select Color box, click on a gray swatch, then click the OK button. 25

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27 Most of the sequences are colored orange, which means they're bacteria. There are also a smaller number of plant sequences the blue nodes and an even smaller number of sequence in the other categories. So coloring by type of life gives a fast, easy way to get an idea of the type of organisms in this sequence set. In the Vizmapper panel, click on Type of Life (indicated by the red arrow in the above image) and select "subgroup 1018 style" to change back to the original family coloring. Now we will search the network for our sequence of interest. Using the search box at the top right of the screen, you can search any type of annotation information available in the network, but you first must configure the search according to the type of annotation information you're interested in. Click on the button indicated by the blue arrow in the above image. 27

28 In the Configure Search Options box, click on Unique Identifier. Scroll down and click on gi. Click the Apply button. In the search box next to the button, type the NCBI GI number for the sequence of interest ( ). Press enter. 28

29 The sequence of interest is highlighted in yellow, and cytoscape automatically zooms in on it. Click on the magnifying glass with the minus sign (indicated by the red arrow in the above image) several times to zoom out. 29

30 In this more global view, the sequence of interest (indicated by the red arrow) clusters with sequences in the dehydrosqualene synthase family (the brown nodes). The squalene synthase family is represented by these green nodes, which are found in a separate cluster. Thus, the sequence of interest appears closer to the dehydrosqualene synthases than the squalene synthases. But we might want to get a better idea of just how close this sequence is to the characterized dehydrosqualene synthases. To do this, we will copy the network and filter it to a more stringent e-value cutoff. Deselect the sequence by clicking anywhere in the white space of the network panel. In the top menu bar, click on File - New - Network - Clone Current Network. 30

31 The Control Panel will now show two copies of the network. You can switch between them by clicking on the name of the desired copy. In the top menu bar, click on Select - Use Old Filters In the Use Filters dialog box, click the Create new filter button. 31

32 In the Filter Creation Dialog box, choose Numeric Filter and click the OK button. Back in the Use Filters box, under "Select graph object of type", select Edge. Under "with a value for numeric attribute" choose -log10(e). Under "that is" choose <, and enter 70 into the box. Click the "Apply selected filter" button. You can see that rather than using BLAST e-value as an edge weight, these networks actually have the -log10 of the E-value as the edge weight. So instead of selecting all edges with an e-value greater than, for example, 1*10-70, you would select all edges with a -log10e-value of less than 70. As the e-value cutoff is made more stringent, there are less connections between nodes that aren t closely related, making it easier to hone in on the group a given sequence is most closely related to. After the filtering process is complete, you'll see all the edges that don't meet this new, more stringent cutoff highlighted in red on the network. 32

33 Delete the edges that don't meet the new e-value cutoff by clicking Edit - Delete Selected Nodes and Edges in the top menu bar. Lay out the network again by clicking on Layout - yfiles - Organic in the top menu bar. 33

34 The larger clusters have now split into smaller, more discrete clusters. Find the sequence of interest in the network by configuring the search box to search by gi and searching for , as described previously. 34

35 Click and drag your mouse to select all sequences in the relevant cluster. The sequence of interest is found within a cluster of sequences from the genus staphylococcus. Not all of them have been assigned to an SFLD family, but the ones that have are members of the dehydrosqualene synthase family. So this network analysis agrees with the previous SFLD analysis though the sequence of interest is annotated as a squalene synthase in Genbank, it appears much more like a dehydrosqualene synthase. 35