MSU SEED Tutorial

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Welcome to SEED Tutorial

Assignment overview

Part I. Find a gene. Locate specific gene/protein page in SEED using protein sequence, ID, or gene name.

Part II. Find and explore relevant genes clusters. Functionally related genes tend to cluster on prokaryotic genomes. In most prokaryotes 50% or more of the genes are clustered with related genes. We call this phenomenon "functional coupling". For any gene that you wish to study, chances are - it will either occur in a cluster, or there will be a corresponding gene in another genome that does occur in a cluster.

Part III. Explore a Subsystem - basics. Identify functional Subsystem(s) in SEED that your protein potentially belongs to, explore a Subsystem page in the browsing mode. Learn basic tools of subsystem visualization and analysis.

Part IV. Annotate a gene. Apply SEED tools (utilizing straightforward homology-based projections, as well as functional and genome context analysis) to reject, refine, or confirm current functional annotation of a protein.

Part V. Explore a Subsystem - advanced. Navigate Subsystem page in the advanced editing mode. Learn to edit existing Subsystems and to encode new ones.

Part VI. Extend a Subsystem. arabidopsisSS.pdf

workshop evaluation form evaluation.pdf

PART 0. Enter SEED using one of these URLs:

http://anno-1.nmpdr.org/public/FIG/index.cgi

http://anno-2.nmpdr.org/public/FIG/index.cgi

http://yersinia.uchicago.edu/public/FIG/index.cgi

Login: collab

Password: bohr.atom

You are on the FIG main (or Search) page now. Please, familiarize yourself with it. You can return to this page from any place in SEED by clicking FIG search link located at the top left corner of every page.

You don't need to authenticate yourself to simply browse the database, but in order to be able to annotate genes or encode Subsystems (Part V and beyond), you'll need a username. Use something in a format master:FirstL, where "FirstL" should be your first name and the first initial of your last name. You can use anything you wish, but do try to make it descriptive and unique. Type your username in the window "User ID" under the caption Searching for Genes or Functional Roles Using Text.

PART I. FIND A GENE

Every protein-encoding gene (PEG) in SEED has an individual WEB page containing a variety of data about the protein and the corresponding gene, tools for protein annotation and analysis, links to external resources, etc.

I.1. Use gene name, protein sequence, EC number, or ID (in GenBank, SwissProt, UniProt, other major databases) to look for the corresponding PEG page in SEED. You can look for a protein of your choice or pick one from the table below. Copy and paste its sequence or ID into an appropriate window on the FIG search page:

(i) If you chose to copy an ID, EC #, or annotation, paste them into the window Searching for Genes or Functional Roles Using Text, and press Search button. To limit your search to a specific genome - highlight this genome and click Search genome selected below button.

(ii) If you chose to copy a sequence, paste it into the window Searching DNA or Protein Sequences (in a selected organism), scroll up the page, highlight the genome of your protein, scroll back down and click Search for matches button (check that Search Program is set for “blastp”)

#	TAIR ID	other IDs	SS	Protein Sequence (fragment) or annotation
1	At1g31860	uni|O82768	+/His	MAVSYNALAQSLARSSCFIPKPYSFRDTKLRSRSNVVFACNDNKNIALQAKVDNLLDRIKWDDKGLAVAIAQNVDTGAVLMQGFVNREALSTTISSRKATFFSRSRSTLWTKGETSNNFINILDVYVDCDRDSIIYLGTPDGPTCHTGEETCYYTSVFDQLNNDEASGNKLALTTLYSLE
2	At3g22425	tr|Q67YN9	+/His	Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19)
3	At4g14910	gi|18414338	+/His	MELLSSSPAQLLRPNLSSRALLPPRTSIASSHPPPPRFLVMNSQSQHRPSISCASPPPGDNGFPAITTASPIESARIGEVKRETKETNVSVKINLDGHGVSDSSTGIPFLDHMLDQLASHGLFDVHVRATGDTHIDDHHTNEDVALAIGTALLKALGERKGINRFGDFTAPLDEALIHVSLDLSGRPYLGYNLEIPTQRVGTYDTQLVEHFFQSLVNTSGMTLHIRQLAGKNSHHIIEATFKAFARALRQATESDPRRGGTIPSSKGVLSRS
4	At4g26900	sp|Q9SZ30	+/His	MEATAAPFSSIVSSRQNFSSSSSIRASSPASLFLSQKSIGNVNRKFKSPRSLSVRASSTSDSVVTLLDYGAGNVRSIRNALRHLGFSIKDVQTPGDILNADRLIFPGVGAFAPAMDVLNRTGMAEALCKYIENDRPFLGICLGLQLLFDSSEENGPVKGLGVIPGIVGRFDASAGIRVPHIGWNALQVGKDSEILDDVGNRHVYFVHSYRAIPSDENKDWISSTCNYGESFISSIRGNV
5	At5g10330	uni|Q9LFT5	+/His	MGVINVQGSPSFSIHSSESNLRKSRALKKPFCSIRNRVYCAQSSSAAVDESKNITMGDSFIRPHLRQLAAYQPILPFEVLSAQLGRKPEDIVKLDANENPYGPPPEVFEALGNMKFPYVYPDPQSRRLRDALAQDSGLESEYILVGCGADELIDLIMRCVLDPGEKIIDCPPTFSMYVFDAAVNGAGVIKVPRNPDFSLNVDRIAEVVELEKPKCIFLTSPNNPDGSIISEDDLLKILEMPILVVLDEAYIEFSGVESRMKWVKKYENLIVLRTFSKRAGLAGLRVGYGAFPLSIIEYLWRAKQPYNVSVAGEVAALAALSNGKYLEDVRDALVRERERLFGLLKEVPFLNPYPSYSNFILCEVTSGMDAKKLKEDLAKMGVMVRHYNSQELKGYVRVSAGKPEHTDVLMECLKFY
6	At4g34740	tr|Q9STG9	+/Pur	Amidophosphoribosyltransferase (EC 2.4.2.14)
7	At1g74260	uni|q9m8d3	+/Pur	MLLQRSSMSQLWGSVRMRTSRLSLNRTKAVSLRCSAQPNKPKAAVSTGSFVTADELPSLVEKPAAEVIHFYRVPLIQESANAELLKAVQTKISNQIVSLTTEQSFNIGLESKLKDEKLSVLKWILQETYEPENLGTDSFLERKKQEGLHAVIVEVGPRLSFTTAWSTNAVSICRACGLDEVTRLERSRRYLLFSKEPLLENQIKEFAAMVHDRMTECVYTQKLVSFETNVVPEEVKYVPVMEKGRKALEEINQEMGLAFDEQDLQYYTRLFREDIKRDPTNVELFDIAQSNSEHSRHWFFAGNMVIDGKPMDKSLMQIVKSTWEANRNNSVIGFKDNSSAIRGFLVNQLRPLLPGSVCLLDVSARDLDILFTAETHNFPCAVAPYPGAETGAGGRIRDTHATGRGSFVVASTSGYCVGNLNMEGSYAPWEDSSFQYPSNLASPLQILIDASNGASDYGNKFGEPMIQGYTRTFGMRLPSG
8	At1g31220	sp|P52422	+/Pur	MESRVLFSSQFNFPVNSPFKTRETSIAPLTPSRNVLSFSFRSPAERCAMRIVPLVKAASSTPQIVAEVDGSSHEPRRKKLAVFVSGGGSNFRKIHEGCSDGSVNGDVVLLVTNKKDCGGAEYARSNGIPVLVFPKAKREPSDGLSPSELVDVLRKYGVDFVLLAGYLKLIPVELVQAFPKRILNIHPALLPAFGGKGLYGIKVHKAVLESGARYSGPTIHFVNEEYDTGRILAQSAVRVIANDTPEELAKRVLHEEHKLYVEVVGAICEERIKWREDGVPLIQNKQNPDEYY
9	At1g03090	gi|17979456	-/Leu	Methylcrotonyl-CoA carboxylase biotin-containing subunit (EC 6.4.1.4)
10	At2g26800	tr|O81027	-/Leu	MQWNGVRRAHSIWCKRLTNNTHLHHPSIPVSHFFTMSSLEEPLSFDKLPSMSTMDRIQRFSSGACRPRDDVGMGHRWIEGRDCTTSNSCIDDDKSFAKESFPWRRHTRKLSEGEHMFRNISFAGRTSTVSGTLRESKSFKEQKYSTFSNENGTSHISNKISKGIPKFVKIVEVGPRDGLQNEKNIVPTSVKVELIQRLVSSGLPVVEATSFVSPKWVPQLADAKDVMDAVNTLDGARLPVLTPNLKGFQAAVSAGAKEVAIFASASESFSLSNINCTIEESLLRYRVVATAAKEHSVPVR
11	At4g34030	sp|Q9LDD8	-/Leu	MLRILGRRVVSASKELTSIQQWRIRPGTDSRPDPFRTFRGLQKGFCVGILPDGVDRNSEAFSSNSIAMEGILSELRSHIKKVLAGGGEEAVKRNRSRNKLLPRERIDRLLDPGSSFLELSQLAGHELYEEPLPSGGIITGIGPIHGRICMFMANDPTVKGGTYYPITIKKHLRAQEIAARCRLPCIYLVDSGGAYLPKQAEVFPDKENFGRVFYNESVMSSDGIPQIAIVLGSCTAGGAY
12	At5g08280	gi|15241573	+/Heme	MDIASSSLSQAHKVVLTRQPSSRVNTCSLGSVSAIGFSLPQISSPALGKCRRKQSSSGFVKACVAVEQKTRTAIIRIGTRGSPLALAQAYETREKLKKKHPELVEDGAIHIEIIKTTGDKILSQPLADIGGKGLFTKEIDEALINGHIDIAVHSMKDVPTYLPEKTILPCNLPREDVRDAFICLTAATLAELPAGSVVGTASLRRKSQILHKYPALHVEENFRGNVQTRLSKLQGGKVQATLLALAGLKRLSMTENVASILSLDEMLPAVAQGAIGIACRTDDDKMATYLASLNHEETRL
13	At3g48730	uni|Q42522	+/Heme	Glutamate-1-semialdehyde aminotransferase (EC 5.4.3.8)
14	At1g69740	sp|Q9SFH9	+/Heme	MATTPIFNASCSFPSTRGIDCKSYIGLRSNVSKVSVASSRIATSQRRNLVVRASESGNGHAKKLGMSDAECEAAVAAGNVPEAPPVPPKPAAPVGTPIIKPLNLSRRPRRNRASPVTRAAFQETDISPANFVYPLFIHEGEEDTPIGAMPGCYRLGWRHGLVQEVAKARAVGVNSIVLFPKVPEALKNSTGDEAYNDNGLVPRTIRLLKDKYPDLIIYTDVALDPYSSDGHDGIVREDGVIMNDETVHQLCKQAVSQARAGADVVSPSDMMDGRVGAIRSALDAEGFQNVSIMSYTAKYASSFYGPFREALDSNPRFGDKKTYQMNPANYREALIEAREDEAEGADILLVKPGLPYLDII
15	At3g27740	uni|O24447	+/Pyr	MAMATRTLGFVLPTSLSSQPSFDRRGGGFRVSVIRCSTSPLTFPTSGVVEKPWTSYNARLVLEDGSIWPAKSFGAPGTRIAELVFNTSLTGYQEILTDPSYAGQFVLMTNPQIGNTGVNPDDEESGQCFLTGLVIRNLSISTSNWRCTKTLADYLTERDIMGVYDLDTRAITRRLREDGSLIGVLSTEQSKTDDELLQMSRSWDIVGIDLISDVSCKSPYEWVDKTNAEWDFNTNSRDGK
16	At1g29900	gi|18397283	+/Pyr	Carbamoyl-phosphate synthase large chain (EC 6.3.5.5)
17	At5g14760	tr|Q94AY1	-/NAD	MAAHVSTGNIHNFYLAGQVYRGQAFSWSSASTFMANPFKEPSWSSGVFKALKAERCGCYSRGISPISETSKPIRAVSVSSSTKYYDFTVIGSGVAGLRYALEVAKQGTVAVITKDEPHESNTNYAQGGVSAVLCPLDSVESHMRDTMVAGAHLCDEETVRVVCTEGPERIRELIAMGASFDHGEDGNLHLAREGGHSHCRIVHAADMTGREIERALLEAVLNDPNISVFKHHFAIDLLTSQDGLNTVCHGVDTLNIKTNEVVRFISKVTLLASGGAGHIYPSTTNPLVATGDGMAMAHRA

Both types of searches should return one or more PEG ID(s) matching your search criteria. A complete PEG ID in SEED looks something like that: fig|562.2.peg.1246, where “fig|562.2” is a genome ID and version, and “peg.1246” is an ID of a specific protein in this genome (the latter are often used in SEED as abbreviated PEG IDs)

Click on the PEG ID to follow the link to the corresponding gene/protein (PEG) page

PART II. Find and explore relevant genes clusters

II.1. We will explore a protein page in detail later. Our strategy in this tutorial is to first show you how to find relevant clusters of genes, by which we mean clusters of functionally related genes that include either the gene you are "positioned on" or a corresponding gene in another organism. The table at the top of a PEG page describes the genes in the region of the chromosome surrounding the gene you are positioned on. The entry for the gene you are positioned on is always shown in green. Just below the table is a small graphical display of the region. The gene you are positioned on is shown here by a green arrow. Genes that are believed to be "functionally related" to it (based on the fact that they occur close to each other in a number of genomes) are shown as blue. Others are red.

Nuclear eukaryotic genes rarely cluster (contrary to the situation in prokaryotes). However, you can still detect related gene clusters – occurring in other genomes – that contain genes homologous to the one you are positioned on. The CL link to the left of the gene leads to the precomputed list of such “indirect” clusters. Try clicking on it. The clusters are sorted by the number of genes in each (Cluster size). PEG IDs in this table are of the close homologs of your query gene (the corresponding similarity score appears in the left-most column). They are linked to the corresponding PEG pages. Open several of them to explore different clusters. Note that even though all these additional clusters are centered around an ortholog of the gene you started with, some of the clusters contain completely different members.

II.2. Explore PINS graphic display that shows the relevant gene clusters in a number of genomes. On every PEG page the columns pins, and fc-sc contain evidence of clustering of the gene you are positioned on with other genes in its immediate neighborhood. The value in the fc-sc column indicates "the strength" of functional coupling and is based on the number of phylogenetically distant organisms in which such clustering is observed. Click on the Pins button just to the left of your gene (shaded green). In a separate window, you should see a portrayal of different versions of the same cluster as they occur in other genomes, “pinned” around homologs of your gene. The query gene is in red. Other homologous genes in the region are shown by arrows with matching colors and numbers. Genes not conserved within the region are colored gray. Mouse over each arrow for more details. Finally, if you choose to click on the Commentary button, another window will pop up containing information about each of the colored sets of homologous genes.

PART III. Explore a Subsystem – the basics.

III.1 Find a Subsystem

There are several ways you may use to find a Subsystem(s) that contains a functional role (assignment) of the query protein:

(i) If a protein has been associated with one (or more) of Subsystems, this will be indicated on the corresponding PEG page by: -- a numerical entry (1, 2, etc) in the SS column of the Context table indicating the number of different Subsystems this PEG is connected to; -- a link on in the Subsystems in which this peg is present table under the Context table.

Note, that activating this link opens a Subsystem page (i) in a simplified “read-only” mode and (ii) with Subsystem spreadsheet display limited to a small number of genomes in the immediate phylogenetic neighborhood of the organism, from the PEG page of which you started. To display all genomes connected to this Subsystem, highlight “Show all” in a drop-down menu and click Show spreadsheet button. To open the same Subsystem in the regular unabbreviated mode – enter it from the FIG main search page.

(ii) If this is not the case, check if any homologs of your protein have been included in a subsystem. Such protein(s) in Similarity table will have a numerical entry (1, 2, etc) in the column In Sub. Go to the respective PEG page (by clicking on its ID) and then follow the subsystem link as described in (i) above.

(iii) You may use the section on the SEED Entry Page: Locate PEGs in Subsystems to search for a relevant subsystem using EC#, functional assignment (if you are lucky), or protein ID (follow instructions on the Entry Page)

(iv) Finally, you may browse a list of subsystems in SEED (or use your browser’s “find in page” functionality) for a potentially relevant term (e.g. NAD biosynthesis, etc). Reach the list of subsystems by clicking on Work on Subsystem button from the FIG main search page.

III.2 Explore a Subsystem

(i) Browse a Subsystem (SS) page. It opens with a Table of Functional Roles constituting this SS. The roles are defined by the most standard descriptive names, for example enzyme names and corresponding Enzyme Classification (EC) numbers, whenever they are available. Note, that role names must exactly match gene annotations in the underlying database. Abbreviations of functional roles are used in Subsystem Spreadsheet below and in SS diagrams.

(ii) Subsets of Roles table. The concept of sub-sets plays an important role in subsystems encoding and interpretation. They usually represent the most compact units, such as multi-subunit complexes, or variants of pathways. A star (*) in front of a sub-set abbreviated name causes all the functional roles grouped in it to collapse into a single column in a Subsystem spreadsheet – a useful feature for displaying synonymic functional roles or subunits of multi-subunit complexes

(iii) Subsystem spreadsheet is a table, in which each column represents a functional role in the subsystem, each row represents a specific genome, and cell are populated with proteins that implement specific functional roles in each organism. Protein IDs in the cells are linked to the corresponding PEG pages.

A small set of tools located immediately under the Subsets of Roles table allows the reduction of spreadsheet display to a selected sub-set of functional roles and/or to a selected group of organisms. The main Subsystem visualization/construction tools are located on SS page below the SS Spreadsheet. Try using the following:

-- limit//expand the number of organisms displayed in the SS Spreadsheet. To display all genomes connected to this Subsystem, highlight “Show all” in a drop-down menu and click Show spreadsheet button (under the Spreadsheet). Use the same menu to limit the display to a specific phylogenetic group

-- show clusters - Check the box near “show clusters” and press Show spreadsheet button. The cells in the same row (genome) highlighted by a matching color contain genes that are located in the immediate vicinity of each other (“clustered”) on the chromosome. Note conserved clusters present in a large number of diverse organisms.

-- variant codes capture meaningful differences in SS implementation across the many genomes. While defining a subsystem, annotators include a collection of functional roles broad enough to cover distinct variations in all relevant organisms. Each subset of functional roles that exists in at least one organism with an operational version of the subsystem constitutes a functional variant. Variant codes allow formal cataloging variations of a pathway implementation across microbial kingdom as well as semi-automatic accurate propagation of gene annotations.

-- [-1] variant code is assigned to all organisms lacking a functional variant of a pathway. By default they are not displayed in a SS spreadsheet. To view these genomes check the “show -1 variants” box and press Show spreadsheet button under the spreadsheet.

-- sorting -- Select an option by_phylo and press update spreadsheet button below. The organisms in the Spreadsheet will be rearranged according to their phylogeny. Selecting “by_pattern” arranges organisms according to the presence/absence of PEGs in the cells of a spreadsheet – a useful tool in analyzing variations in SS implementation in different organisms

(iv) Subsystem diagram (graphic representation of a pathway) is often helpful in analyzing a SS and assigning variant codes. These graphic maps (available for a number of SSs) can be accessed via a link above a SS Spreadsheet. Functional roles are shown by abbreviations in boxes. Key metabolites (precursors, products) and intermediates are shown by abbreviations or roman numerals in circles (linked to the KEGG Compounds db). Diagrams can be highlighted to show the presence/absence of genes implementing each functional role in a specific organism by activating the Color links located in the SS Spreadsheet in the Genome ID column.

(iv) Notes section at the bottom of each SS page contains annotator’s comments, lists open problems identified during SS encoding and analysis, and - most importantly - explains variant codes identified in the SS.

QUIZ: You have 10 minutes to explore a hypothetical protein in SEED. What can you tell about it?

Help with navigating PEG pages in SEED is available here: PEG page Help.

Arabidopsis thaliana “mystery proteins” for investigation

PART IV. Gene annotation in SEED.

SEED provides a very rich annotation environment. Just a few of the many possible strategies to annotate a gene in SEED are described below. Choose one of these strategies to annotate your protein of interest or one of the proteins from the table below. In order to choose a proper annotation for your protein:

- analyze annotations of the same protein in other databases, open and explore Similarity table, and/or follow Show Abstract Coupling and CL (show related clusters) links;

- keep in mind that protein annotations in SEED also serve as their only connections to Subsystems. In order for a protein to be associated with a Subsystem, it’s annotation should exactly match the name of the corresponding functional role.

Before annotating - please, check whether any close homologs of your protein in other organisms have been connected to a Subsystem already. If you believe that your protein belongs to the same Subsystem, we strongly encourage you to use the same annotation. However, if you consider it completely wrong – the right thing to do will be to contact the Subsystem curator and discuss the issue with him or her.

Annotating from a PEG page

1. Annotating using the Assignments for Essentially Identical Proteins table:

-- if you click on one of the blue arrows under the ASSIGN column, the current assignment of the protein you are position on is changed to match that of the row in which you clicked

2. Annotating using To Make an Annotation link

-- when this link is activated, a form for PEG annotation is displayed

-- type the new gene assignment into New Function window and press add annotation button (note that to avoid typos, it is better to copy the corresponding functional role from your SS and paste it into this window)

-- the New Annotation window can be used to add references and comments (these data are not displayed on a PEG page)

-- a report is generated in a new window – it is safe to close it

3. Annotating using Similarities table

-- Generate a non-redundant list of similarities:

-- request between 150 and 350 homologs by typing the corresponding number in the “Max sims” window

-- choose Just FIG IDs (all) from the drop-down menu

-- click Similarities button.

-- Analyze the resultant table of homologs, find a homolog with specific annotation you want to propagate

-- Click on a round check-box in the ASSIGN from column near a homolog with annotation you want to propagate

-- Make sure ASSIGN to/SELECT current PEG check-box and ASSIGN/annotate with form check-box above the SIMs table are checked

-- press assign/annotate button

-- the current PEG is re-annotated, a report page is generated in a new window - it is safe to close it

-- note that the same annotation can also be propagated to any number of homologs in the Similarities table by checking the corresponding square check-boxs in the left-most ASSIGN to//SELECT column.

Annotating from a Commentary page

The unique advantage of this strategy over straight-forward homology projections is in considering chromosomal context: only close homologs in similar chromosomal clusters are assigned with identical specific functions.

-- use To Compare Region or Pins tools to generate Commentary page as described in PEG page Help.

-- in the PEG column check the PEG you intend to re-annotate

--chose “Function” to annotate it from: you can use either Function or Uniprot Function columns. Check the box near the function you want to propagate

-- press assign/annotate button

-- your PEG will be re-annotated to match the source you selected

-- close report page

Try annotating these proteins

If you got that far – CONGRATULATIONS!

You are a certified SEED user!