Protein 3D-structure analysis

Exercises

PracticalsFind 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

PracticalsHow 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.htmlhttp://www.rcsb.org/pdb/statistics/contentGrowthChart.do?content=total&seqid=100

PracticalsHow many structures determined by X-ray crystallography, NMR, Electron microscopy?

Use Advanced Query at RCSB PDB

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&maxLabel=5&numOfbars=10&name=Resolution

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

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

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 p38Most easily done via the UniProt entry

Find the UniProt AC for p38.

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

Identifiy the residues that form hydrogen bonds with the product, via PDBsum and from the RCSB PDB website: e.g. 2IJMhttp://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/RunLigplot.pdf?pdb=2ijm&pdf=YES&file=ligplot02_01and Ligand Explorer from RCSB PDB (shown below)

At RCSB PDB, find structures for proteins similar to p38Query: p38 imatinib in main search window, choose 3HEC:You get kinases

find structures for proteins similar to p38via 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

Sequence: http://www.uniprot.org/uniprot/O00444.fasta

Use the sequence of human PLK4 to find a matching structure (advanced search tools)

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

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

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

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 DIOXYGENASETaxons: Mammalia (alkB homologs), and bacteria (alkB from E.coli)

What are their ligands?Answer: alpha-ketoglutarate and ironhttp://www.rcsb.org/pdb/explore.do?structureId=2IUW

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 explorerhttp://www.uniprot.org/uniprot/Q9C0B1#section_featureshttp://www.uniprot.org/uniprot/P05050#section_features

B) Get an alignment from RCSB PDB or DALIhttp://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

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 differenthttp://www.uniprot.org/uniprot/P05050#section_x-refhttp://www.uniprot.org/uniprot/Q9C0B1#section_x-ref

Find 3D-structures for transthyretin at RCSB PDB, then look for proteins with similar 3D-structurehttp://www.rcsb.org/pdb/explore/explore.do?structureId=1THCSimilar 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 searchhttp://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.c.d.e.b.b.htmlhttp://www.cathdb.info/cathnode/2.60.40.180NB: the proteins are too similar to be distinguished by these means

More things to do (facultative)

Find protein structures with aspirin as ligandFind the chemical structure for aspirinGet all structures with the ligand aspirin. How many are there?-Go to CHEBI, get Smiles for aspirin-http://www.ebi.ac.uk/chebi/advancedSearchFT.do-http://www.ebi.ac.uk/chebi/searchId.do;E5D46C3E2363F473FCE4C201CC2078A6?chebiId=CHEBI:15365

-Go to RCSB PDB, use advanced search (smiles)-You can also try and draw “aspirin” using CHEBI