1. Protein 3D-structure analysis Exercises

2. Practicals Find update frequency for RCSB PDB: weekly. When was the last update? How many protein structures are there in wwPDB? http://www.rcsb.org/pdb/home/home.do http://www.ebi.ac.uk/pdbe/#m=0&h=0&e=0&r=0&l=0&a=0&w=0

3. Practicals How many NEW structures were deposited in 2010 vs 2011? Hint: try this at PDBe vs RCSB PDB Much easier at RCSB PDB: http://www.rcsb.org/pdb/static.do?p=general_information/pdb_statistics/index.html http://www.rcsb.org/pdb/statistics/contentGrowthChart.do?content=total&seqid=100

4. Practicals How many structures determined by X-ray crystallography, NMR, Electron microscopy? Use Advanced Query at RCSB PDB

5. Find the structure with the highest resolution at RCSB PDB http://www.rcsb.org/pdb/statistics/histogram.do?mdcat=refine&mditem=ls_d_res_high&minLabel=0&m axLabel=5&numOfbars=10&name=Resolution

6. How many structures determined by X-ray crystallography, NMR, Electron microscopy? Use advanced Query at PDBe http://www.ebi.ac.uk/pdbe-srv/view/search?search_type=advanced

7. At RCSB PDB, find structures with imatinib as ligand. How many structures contain this ligand? What type of proteins contain this ligand? Use advanced query. Verify using the PDB ID for imatinib=STI Answer: Protein kinases

8. Amongst proteins with bound imatinib, there are structures for p38. http://www.rcsb.org/pdb/explore/explore.do?structureId=3HEC Find the official protein and gene name of p38 Most easily done via the UniProt entry Find the UniProt AC for p38.

9. Find structures for a human protein kinase with bound ADP and a resolution between 2 and 2.5 A: Use main query window, find structures with ligand ADP, then use options (Species, Resolution, Enzyme classification, …) to filter the result

11. Identifiy the residues that form hydrogen bonds with the product, via PDBsum and from the RCSB PDB website: e.g. 2IJM http://www.ebi.ac.uk/thornton-srv/databases/cgi- bin/pdbsum/RunLigplot.pdf?pdb=2ijm&pdf=YES&file=ligplot02_01 and Ligand Explorer from RCSB PDB (shown below)

12. At RCSB PDB, find structures for proteins similar to p38 Query: p38 imatinib in main search window, choose 3HEC: You get kinases

13. find structures for proteins similar to p38 via the DALI database (FSSP link on PDBsum page). What kind of proteins do you recover? http://ekhidna.biocenter.helsinki.fi/dali_server/results/3hec/index.html

14. Sequence: http://www.uniprot.org/uniprot/O00444.fasta Use the sequence of human PLK4 to find a matching structure (advanced search tools)

15. Get sequence: http://www.uniprot.org/uniprot/O00444.fasta

16. Look at protein structure classification in CATH and SCOP (e.g. via links for 3BRB from PDB or PDBsum). The aim is essentially to see that there are quantitative means to classify domain structures No hit in SCOP, so try keyword search, or start at top of hierarchy http://scop.mrc- lmb.cam.ac.uk/scop/search.cgi?ver=1.75&key=kinase&search_type=scop

17. How many PDB structures are represented in CATH and in SCOP? What is the reason for the difference relative to the total number of experimental structures? Check their home pages: http://www.cathdb.info/ http://scop.mrc-lmb.cam.ac.uk/scop/index.html More automated procedures can handle more data – human beings can handle less data, but using your brains adds extra value

18. Starting with the structure for human FTO, find other proteins with similar 3D-structure via PDB http://www.rcsb.org/pdb/explore/structureCluster.do?structureId=3LFM or DALI (FSSP) : http://ekhidna.biocenter.helsinki.fi/dali_server/results/3lfm/index.html What kind of proteins do you find (function, taxonomy)? Answer: dioxygenases, more specifically ALPHA-KETOGLUTARATE- DEPENDENT DIOXYGENASE Taxons: Mammalia (alkB homologs), and bacteria (alkB from E.coli) What are their ligands? Answer: alpha-ketoglutarate and iron http://www.rcsb.org/pdb/explore.do?structureId=2IUW

19. Do an alignment based on the 3D-structure and check if the ligand- binding residues are conserved. Do this with entries from closely related/different taxons. A)Get the ligand-binding sites using PDBsum, UniProt annotation, or Ligand explorer http://www.uniprot.org/uniprot/Q9C0B1#section_features http://www.uniprot.org/uniprot/P05050#section_features B) Get an alignment from RCSB PDB or DALI http://www.rcsb.org/pdb/workbench/showPrecalcAlignment.do?action= pw_fatcat&mol=3LFM.A&mol=2IUW.A Very low sequence identity, but secondary structure is almost the same. Binding sites for catalytic iron (HxD and H) are boxed

20. Can you get the same proteins, doing a normal BLAST search with FTO? Answer: No, they are too different Have a look at the domain structure (InterPro) of FTO and the proteins that you identified. Compare InterPro lines: they are different http://www.uniprot.org/uniprot/P05050#section_x-ref http://www.uniprot.org/uniprot/Q9C0B1#section_x-ref

21. Find 3D-structures for transthyretin at RCSB PDB, then look for proteins with similar 3D-structure http://www.rcsb.org/pdb/explore/explore.do?structureId=1THC Similar structure: 2H6U What is the function of transthyretin, respectively of other proteins with similar structure? Have a look at the corresponding UniProt entries! How are they classified at CATH / at SCOP? Use text search http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.c.d.e.b.b.html http://www.cathdb.info/cathnode/2.60.40.180 NB: the proteins are too similar to be distinguished by these means

22. More things to do (facultative) Find protein structures with aspirin as ligand Find the chemical structure for aspirin Get all structures with the ligand aspirin. How many are there? -Go to CHEBI, get Smiles for aspirin -http://www.ebi.ac.uk/chebi/advancedSearchFT.dohttp://www.ebi.ac.uk/chebi/advancedSearchFT.do -http://www.ebi.ac.uk/chebi/searchId.do;E5D46C3E2363F473FCE4C2 01CC2078A6?chebiId=CHEBI:15365http://www.ebi.ac.uk/chebi/searchId.do;E5D46C3E2363F473FCE4C2 01CC2078A6?chebiId=CHEBI:15365 -Go to RCSB PDB, use advanced search (smiles) -You can also try and draw “aspirin” using CHEBI