Nitrogen Fixing GO Annotations

UW Fall 2013 Example

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)).

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! ***

Step 1. Suitable paper…

Click on this, or try UW online library

or go to the library

****** NEVER pay for an article!! *******

PMID:12045829

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!

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

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

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

proteins!

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)

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! ***

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

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

Step 6: Filling in a GO annotation for A. brasilense NtrY on GONUTS using PMID:12045829

QualifierGO IDReferenceEvidence codeWith/fromNotes

- (almost never needed)GO:0005886PMID:12045829ISM: Inferred from Sequence ModelPMID:7108955Fig. 2B

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

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)).

Are there more annotations in PMID:12045829?

Maybe!!

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 caulinodansCc = Caulobacter crescentusSm = Sinorhizobium melilotiMl = Mesorhizobium loti

Figure 4. No change/doesn’t complement == nothing to annotate

Table 2. No change/doesn’t complement == nothing to annotate

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.

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)).

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)