1. Nitrogen Fixing GO Annotations UW Fall 2013 Example

2. 6 parts to making functional annotations Find a suitable article on PubMed about a protein Find the same protein in UniProt & get the accession # Use UniProt accession to make an editable protein page on GONUTS Find a suitable GO term based on figure(s) &/or table(s) characterizing protein Pick a suitable evidence code based on how the protein was characterized Enter (and save) your GO annotation on the protein’s page in GONUTS complete with the notes filled in (figure(s)/table(s)).

3. Step 1. Find a suitable paper in PubMed about a protein involved in nitrogen fixation nitrogen fixation AND protein purification AND characterization *** No review articles! ***

4. Step 1. Suitable paper… Click on this, or try UW online library or go to the library ****** NEVER pay for an article!! ******* PMID:12045829

5. Step 2: Can we find those proteins in UniProt? PMID:12045829 is about Azospirillium brasilense NtrY and NtrX – G8AQ26 = A. brasilense NtrX – P45675 = A. brasilense NtrY Has this paper been annotated already on their protein pages on UniProt? – No!

6. Step 3: Make the pages for the proteins in GONUTS where we can annotate - http://gonuts.tamu.edu

7. Step 3: Protein pages in GONUTS Has this paper been annotated already on their protein pages on GONUTS (i.e. by another student)? – No PMID:12045829 doesn’t show up in the “Reference” column of the Annotation table

8. Figure 1. Not suitable for GO annotations – no experimental/computational analysis about proteins!

9. Figure 2. GO Annotation with evidence code “Inferred from Sequence Model” for NtrY Authors show a hydropathy plot for NtrY in Fig 2B – hydropathic regions predicted to span plasma membrane Based on a sequence model from Kyte & Doolittle (1982)

10. Step 4: What would be a suitable GO term for the annotation then? GO:0005886 – plasma membrane – Ok… but is there a better one? GO:0005887 – integral to plasma membrane – Perfect! *** Don’t forget – never annotate to “binding” terms or “response to” terms for CACAO! ***

11. Evidence Codes for CACAO Evidence codes describe the type of work or analysis done by the authors  IDA: Inferred from Direct Assay  IMP: Inferred from Mutant Phenotype  IGI: Inferred from Genetic Interaction  ISO: Inferred from Sequence Orthology  ISA: Inferred from Sequence Alignment  ISM: Inferred from Sequence Model  IGC: Inferred from Genomic Context If it’s not one of these 7, your annotation is incorrect!!! http://gowiki.tamu.edu/wiki/index.php/evidence_codes

12. Step 5: Choose the appropriate evidence code *** ISM needs something in the with/from field *** ISM – PMID of paper describing the sequence modeling program – Kyte & Doolittle, 1982 paper PMID:7108955

13. Step 6: Filling in a GO annotation for A. brasilense NtrY on GONUTS using PMID:12045829 Qualifier GO ID Reference Evidence code With/from Notes - (almost never needed) GO:0005886 PMID:12045829 ISM: Inferred from Sequence Model PMID:7108955 Fig. 2B

14. CACAO Competition For each complete & correct GO annotation: – 5 points and – credit for 1 acceptable annotation 5 rounds – EACH ROUND = 1 week to enter GO annotations, 1 week to challenge annotations made by other teams Challenges let you steal: – Points identifying problems suggesting appropriate fixes – Credit for fixing all problems with an annotation

15. 6 parts to making functional annotations Find a suitable article on PubMed about a protein Find the same protein in UniProt & get the accession # Use UniProt accession to make an editable protein page on GONUTS Find a suitable GO term based on figure(s) &/or table(s) characterizing protein Pick a suitable evidence code based on how the protein was characterized Enter (and save) your GO annotation on the protein’s page in GONUTS complete with the notes filled in (figure(s)/table(s)).

16. Are there more annotations in PMID:12045829? Maybe!!

17. Figure 3. GO Annotation for NtrX or NtrY with evidence code “Inferred from Sequence Alignment”, but only if one of the aligned proteins is experimentally characterized to an appropriate GO term!! For time reasons, I am not going to go through this annotation. It might be possible to get an annotation for NtrX and/or NtrY if this works. Ac = Azorhizobium caulinodans Cc = Caulobacter crescentus Sm = Sinorhizobium meliloti Ml = Mesorhizobium loti Figure 4. No change/doesn’t complement == nothing to annotate Table 2. No change/doesn’t complement == nothing to annotate

18. Figure 5. Complementation of nifR3ntrBC A. brasilense mutant could get you an annotation using IGI: Inferred from Genetic Interaction for each of NtrX and NtrY GO term – GO:0042128 nitrate assimilation Reference – PMID:12045829 Evidence code – IGI: Inferred from Genetic Interaction – REQUIRES YOU TO FILL IN with/from with/from – UniProt accessions from A. brasilense for: NtrX or NtrY (expressing both from plasmid) NifR3 – P45672 NtrB – P45642 NtrC – P45671 Notes – Figure 5.

19. 6 parts to making functional annotations Find a suitable article on PubMed about a protein Find the same protein in UniProt & get the accession # Use UniProt accession to make an editable protein page on GONUTS Find a suitable GO term based on figure(s) &/or table(s) characterizing protein Pick a suitable evidence code based on how the protein was characterized Enter (and save) your GO annotation on the protein’s page in GONUTS complete with the notes filled in (figure(s)/table(s)).

20. What else can we do with PMID:12045829? Look at this paper’s references!! – Lots of papers mentioned in intro that you can go find & annotate – Nicely written up here (easier to find GO terms)