The server uses Position Specific Scoring Matrices (PSSM) to calculate the level of conservation for each residue in the protein. The PSSM is read by the program and it calculates, at each position how often the residue is conserved in the sequence of homologous proteins.
Once the frequency of conserved residues amongst homologues is obtained from the PSSM, the server normalises the frequencies and replaces the B temperature factor field from the Protein Data Bank (PDB) file corresponding to the query. It then displays the modified PDB file using the JMol Java Applet, colouring the residues according to the temperature that has been replaced by the residue conservation, "Et voila!".
There are two possibilities.
If the protein has been published in the Protein Data Bank and you know its identifier and the chain you want to look at, just enter the PDB id and the chain ID in the corresponding fields. The server will retrieve the precalculated PSSM and the PDB file from our database and will quickly return the result.
If, on the other hand the structure you want to look at is not in the PDB database but you have the coordinates file. You can upload the file. The server will extract the sequence, calculate the PSSM and generate the display. The second option takes longer, as the PSSM calculation involves a search in our protein sequences database. During the calculation, a waiting page will appear with a progress bar, the waiting page will update itself automatically every 30 seconds, until calculation is finished.
This colour gradient from blue to red, is the respresentation of the degree of residue conservation in the protein sequence. A blue residue means that it is not very well conserved amongst homologous sequences; the more intense blue the less conserved. Conversely, red residues are very well conserved, the more intense red the more conserved. A white residue, means that it is only conserved in half of homologues.
| Non Conserved | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | _ | Conserved |
Residue conservation is a very useful type of information, as it can reveal the importance of a given residue in the structure, from a structural or functional point of view.
From a structural point of view, a very well conserved residue, might be important for the correct folding of the protein or for maintaining the stability of the structure. Generally, residues buried in the core of the protein tend to be more conserved.
From a functional point of view, conserved residues, in particular if present on the surface of the protein, are likely to be responsible for the function of the protein; for example an active site. A patch of conserved residues on the surface of the molecule, especially if the residues are hydrophobic or charged might be the interface, for interaction with other proteins. Also, in proteins involved in molecular recognition, like antibodies, the structure is usually very well conserved, but the residues responsible for specific recognition have a lot of variability; an antigen recognition surface for example.
The PSSM is a form of profile, derived from a multiple sequence alignment. PSSM are the intermediates profiles used by the program PSI-BLAST (Altschul et al.) for searching homologues in a protein sequence database. To calculate the PSSM, the server executes three iterations of a PSI-BLAST search against a database of protein sequences. Our database of sequences, currently contains, all sequences of proteins of known structures that are present in the PDB (minus redundant sequences) and all the sequences from the NCBI nr database of non redundant sequences from sequenced genomes.
In some cases, if the structure has been in the database for a long time, the PSSM may be out of date or precisely, homologous sequences have been added to the database but the PSSM has not been updated accordingly. In this case, to have a more up to date conservation data, it is preferable to recalculate the PSSM. On the other hand, this is a slower option than using the precalculated PSSM.
Yes, all you need is to fill the PDB id field from the form with the comma separated list of chains you want to visualise. If you want to see all the chains, just leave the field empty.
Don't worry, just be happy; the server can deal with that. Just leave the PDB id field in the form empty.
The server will read the MODRES field from the PDB file and replace the modified residue with its original equivalent (as coded in the gene).
The PSSM are calculated from protein sequences, using PSI-BLAST, therefore Nucleotide Sequences are ignored.
In the result page, click on the "Download PDB file" link. Your browser should prompt you, to save the file on your disk or start any application associated with PDB file format. The conservation score is stored in the B-factor field of the PDB file. You can visualise the results with any molecular viewer that can open PDB file and color the structure according to the temperature factor.
Our computing resources are somewhat limited, so please be considered to other users. If you need to submit more than 10 structures or force PSSM calculation within an hour, please contact us beforehand at the following address: .
Bear in mind that when you submit a single structure, this will use as many processors on our system as there are chains in the structure.
Yes, but to save space on our system, the results are deleted after two weeks and the link will not be valid anymore. If you want to keep your result, you should download the PDB file from the link provided.
It is the percentage of occurence of the residue at this position amongst homologues of the sequence
Yes, when you click on the residue in the sequence you will see a bar chart with the relative frequency of the 20 residues at this position in the sequence family.