1. Welcome to the Protein Database Tutorial. This tutorial will describe how to navigate the section of Gramene that provides collective information on proteins from rice (Oryza sp.). The protein entries are annotated by associating the following three concepts of Gene Ontology (GO):Gene Ontology (GO) Molecular function of the gene product. Biological process in which the gene product is involved. Cellular component where the gene product is localized.

2. Searching the Protein Database 1. Search by entering a term. 2. Search by Pfam or PROSITE (See Slide 9 for more info). 3. Browse by Gene Ontology (See Slide 12 for more info). or 4. Search by sequence homology (See Slide 15 for more info). or Click at HELP on the protein search section for more information

3. Select “Wildcard” for a broader search. Describes the type of experiments carried out to ascertain the protein’s function, e.g. ISS: Inferred from sequence or structural similarity. Select to view the Protein General Information Page. Search by either a protein or gene name, a subspecies, a cultivar/germplasm, the Gramene/Swissprot Accession number or the Swissprot ID. Results for term search

4. If the protein is an enzyme and has a recognized Enzyme Commission (EC) number, select to view more information about the enzyme. Recognized symbol for the gene encoding this protein. Select to view the location of the gene in the rice genome (Genome Browser tutorial available also). Cross references to GenBank and SWISSPROT protein entries. Select to retrieve the sequence in FASTA format. Protein General Information Page

5. Select to view more information on the function, process, or cellular component association from the Gene Ontology (GO) Database (Ontology tutorial available also). Select to view the reference source used to assign the GO term. Next view in Protein: Gene ontology associations

6. In order to find the homologs from the already selected species, select to link to the National Center for Biotechnology Information’s (NCBI) BLink display. By default the views show the alignments by taxonomic proximity and not BLAST score (BLAST tutorial available also). In order to find the structural homologs, select to link to the NCBI’s PDB and Univ. College London’s SAS database of protein structures. Since there is a limitation on structural homologs, it is not uncommon to have no hits. Next view in Protein: Similarity to other proteins

7. Identifies the Protein family. Select to view more details from Pfam/PROSITE. Select to view a list of all the protein entries that belong to this Protein family or share the PROSITE domain. Select to view physiochemical properties at ProtParam. Identifies any important features found on the protein molecule, the feature start and stop position, and the reference and experiment type used in ascertaining the location and type of feature. Next view in Protein: Associated features

8. Select to search for more references in the literature database that were not cited in the curation (Literature tutorial available also). Final view in Protein: Literature references

9. Search by either a Pfam / PROSITE Class or an identifier such as PF00281. Note: Some proteins do not have associations to Pfam and PROSITE features. This will restrict your search. Search by Pfam or PROSITE

10. Actual name of the Pfam/PROSITE Class. Pfam/PROSITE Accession. Select to display all the protein entries that are either members of that Protein Family or carry that PROSITE recognized domain. Results for Pfam or PROSITE Search

11. Protein Name as recorded in Gramene. Select to view the Gramene Protein General Information Page. Select to view the SWISSPROT protein entry page. Select to view the GenBank (NCBI) protein entry page. Select to view the Pfam/PROSITE annotation. Select to view the Pfam/PROSITE database for more information on the protein family or the Domain. Proteins associated to a given Pfam or PROSITE class

12. Selecting the terms allows you to view an Ontology database which displays a list of proteins associated with these particular words/concepts or their children (Ontology tutorial available also). Browse by Gene Ontology (GO) Note: Some proteins do not have associations to Pfam and PROSITE features. This will restrict your search.

13. The term’s lineage in a molecular function category tree and its children. Number of proteins associated with this term. Expandable tree Select to download/display a list of proteins that were annotated to this function. Results for Gene Ontology (GO)

14. Column headings allow for alphabetical sorting of the list. Select the protein/gene name to view the Gramene Protein General Information Page. Children terms in the tree following the Primary vocabulary term for which the protein function was annotated. Results for Gene Ontology (GO)

15. 1. Paste desired sequence in either FASTA or raw format. 2. Select the Program “blastp.” 3. Select Rice_Swissprot database or Rice_japonica_Genemodel_Prtoteins 4. Select “Run BLAST.” 5. On the BLAST results page, select the accession that gives you the best hit. This will link you to the respective Protein General Information Page. Search by sequence homology to proteins (BLAST tutorial available also.)

16. Thank you for using this tutorial. We appreciate any comments or suggestions. Please click here to send your feedback. http://www.gramene.org/db/feedback/send_feedback